本申请是申请日为2008年10月14日、中国申请号为200880111668.6、发明名称为“与IL-4和/或IL-13结合的抗体及其用途”的发明申请的分案申请。This application is a divisional application of an invention application with a filing date of October 14, 2008, a Chinese application number of 200880111668.6, and an invention title of "antibodies that bind to IL-4 and/or IL-13 and uses thereof".
发明领域field of invention
本发明涉及新颖的抗IL-4抗体、抗IL-13抗体以及双特异抗IL-4/抗IL-13抗体及其在因不当的IL-4和/或IL-13活性或代谢而造成的哺乳动物包括人的疾病或障碍的改善、治疗和预防方面的用途。感兴趣的抗体可能阻断配体如IL-4或IL-13与受体或受体复合物如IL-4Rα、IL-13Rα1和IL-13Rα2的结合和/或信号传导。本发明还披露了含有感兴趣抗体的预防、免疫治疗和诊断组合物以及它们在预防或治疗哺乳动物包括人的疾病的方法中的用途，这些疾病是由淋巴样细胞和非淋巴样细胞，包括单核细胞、成纤维细胞和内皮细胞的不适当代谢和/或活性而引起的。这些疾病包括自身免疫缺陷以及由炎症引起或以炎症为特征的疾病，如变应性哮喘和皮炎。The present invention relates to novel anti-IL-4 antibodies, anti-IL-13 antibodies and bispecific anti-IL-4/anti-IL-13 antibodies and their role in inappropriate IL-4 and/or IL-13 activity or metabolism. Use in the improvement, treatment and prevention of diseases or disorders in mammals including humans. Antibodies of interest may block binding and/or signaling of ligands such as IL-4 or IL-13 to receptors or receptor complexes such as IL-4Rα, IL-13Rα1 and IL-13Rα2. The invention also discloses prophylactic, immunotherapeutic and diagnostic compositions containing antibodies of interest and their use in methods of preventing or treating diseases in mammals, including humans, caused by lymphoid and non-lymphoid cells, including Inappropriate metabolism and/or activity of monocytes, fibroblasts and endothelial cells. These diseases include autoimmune deficiencies as well as diseases caused by or characterized by inflammation, such as allergic asthma and dermatitis.
发明背景Background of the invention
白介素-4(IL-4)是一种对于淋巴B和T细胞以及许多非淋巴样细胞，包括单核细胞、内皮细胞和成纤维细胞具有广谱生物效应的多效性细胞因子。例如，IL-4刺激几个依赖于IL-2和IL-3的细胞系的增殖，诱导II类主要组织相容性复合体在静息B细胞上的表达，并增强人B细胞的IgG4和IgE分泌。IL-4与Th2-型的免疫响应有关，是由Th2细胞产生的，并促进Th2细胞的分化。多种障碍都牵涉到IL-4，如过敏和哮喘。Interleukin-4 (IL-4) is a pleiotropic cytokine with a broad spectrum of biological effects on lymphoid B and T cells and many nonlymphoid cells, including monocytes, endothelial cells and fibroblasts. For example, IL-4 stimulates the proliferation of several IL-2- and IL-3-dependent cell lines, induces the expression of major histocompatibility complex class II on resting B cells, and enhances IgG4 and IgE secretion. IL-4 is associated with Th2-type immune responses, is produced by Th2 cells, and promotes the differentiation of Th2 cells. IL-4 has been implicated in a variety of disorders, such as allergies and asthma.
IL-13是最近发现的由活化T淋巴细胞、B淋巴细胞以及活化后的肥大细胞分泌的112个氨基酸的细胞因子(Minty,A.等人,Nature,1993,362,248-250,和McKenzie,A.N.等人,Proc.Natl.Acad.Sci.U.S.A,1993,90,3735-3739)。IL-13 is a recently discovered cytokine of 112 amino acids secreted by activated T lymphocytes, B lymphocytes, and activated mast cells (Minty, A. et al., Nature, 1993, 362, 248-250, and McKenzie, A.N. et al., Proc. Natl. Acad. Sci. U.S.A, 1993, 90, 3735-3739).
由于和IL-4具有许多共同的生物特性，IL-13被描述为一个IL-4样的细胞因子。在B细胞(Defrance,T.等人,J.Exp.Med.,1994,179,135-143,Punnonen,J.等人,Proc.Natl.Acad.Sci.(USA),1993,90,3730-3734,Fior,R.等人,Eur.Cytokine Network,1994,5,593-600)、单核细胞(Muzio,M.R.F.等人,Blood,1994,83,1738-1743,De WaalMalefyt,R.等人,J.Immunol,1993,151,6370-6381,Doyle,A.等人,Eur.J.Immunol.1994,24,1441-1445,Montaner,L.J.等人,J.Exp.Med.,1993,178,743-747,Sozzani,P.等人,J.Biol.Chem.,1995,270,5084-5088)以及其它非造血细胞(Herbert,J.M.等人,FebsLett.,1993,328,268-270,和Derocq,J.M.等人,Febs Lett.1994,343,32-36)上，其活性确实类似于IL-4的活性。另一方面，与IL-4相反，它对静息或活化的T细胞并没有特异效应(Zurawuki,G.等人,Immunol.Today,1994,15,19-26)。Since it shares many biological properties with IL-4, IL-13 has been described as an IL-4-like cytokine. In B cells (Defrance, T. et al., J. Exp. Med., 1994, 179, 135-143, Punnonen, J. et al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 3730-3734 , Fior, R. et al., Eur.Cytokine Network, 1994,5,593-600), monocytes (Muzio, M.R.F. et al., Blood, 1994,83,1738-1743, De WaalMalefyt, R. et al., J. Immunol, 1993, 151, 6370-6381, Doyle, A. et al., Eur.J. Immunol.1994, 24, 1441-1445, Montaner, L.J. et al., J.Exp.Med., 1993, 178, 743-747, Sozzani, P. et al., J.Biol.Chem., 1995, 270, 5084-5088) and other non-hematopoietic cells (Herbert, J.M. et al., FebsLett., 1993, 328, 268-270, and Derocq, J.M. et al., On Febs Lett. 1994, 343, 32-36), its activity is indeed similar to that of IL-4. On the other hand, in contrast to IL-4, it has no specific effect on resting or activated T cells (Zurawuki, G. et al., Immunol. Today, 1994, 15, 19-26).
A.J.Minty以及关于IL-13的评论文章详细地描述了IL-13在单核细胞/巨噬细胞、B淋巴细胞以及有些造血前体上的多种生物活性。此外，还有几份数据表明，这一细胞因子在其它细胞类型上具有多效效应。这些直接受到IL-13影响的非造血细胞为内皮细胞、小胶质细胞、角质形成细胞以及肾和结肠癌。A.J. Minty and review articles on IL-13 describe in detail the various biological activities of IL-13 on monocytes/macrophages, B lymphocytes and some hematopoietic precursors. In addition, there are several data showing that this cytokine has pleiotropic effects on other cell types. These non-hematopoietic cells directly affected by IL-13 are endothelial cells, microglia, keratinocytes, and renal and colon cancers.
细胞内生物分子所发射信号的分析阶段之一是识别其膜受体。针对IL-13受体的研究表明，IL-13和IL-4有共同的受体，或者至少具有共同受体复合物的一些组分，以及共同的信号转导元素(Zurawski S.M.等人,Embo Journal,1993,12,2663-2670,Aversa,G.等人,J.Exp.Med.,1993,178,2213-2218,Vita,N.等人,Biol.Chem.,1995,270,3512-3517,Lefort,S.等人,Febs Lett.,1995,366,122-126)。这一受体存在于多种类型细胞的表面，其数目随所考虑细胞类型不同而有变化。A.J.Minty指出了IL-13和IL-4受体的相当的分布(Interleukin-13for Cytokines in Health and Disease(健康和疾病中的细胞因子白介素-13).编著D.G.Remick和J.S.Frie,Marcel Decker,N.Y.1996)。One of the stages in the analysis of signals emitted by intracellular biomolecules is the identification of their membrane receptors. Studies targeting the IL-13 receptor have shown that IL-13 and IL-4 share a common receptor, or at least some components of a common receptor complex, as well as common signal transduction elements (Zurawski S.M. et al., Embo. Journal, 1993, 12, 2663-2670, Aversa, G. et al., J. Exp. Med., 1993, 178, 2213-2218, Vita, N. et al., Biol. Chem., 1995, 270, 3512- 3517, Lefort, S. et al., Febs Lett., 1995, 366, 122-126). This receptor is present on the surface of many types of cells, the number of which varies with the cell type considered. Comparable distribution of IL-13 and IL-4 receptors was pointed out by A.J. Minty (Interleukin-13 for Cytokines in Health and Disease). Eds. D.G. Remick and J.S. Frie, Marcel Decker, N.Y. 1996).
细胞表面受体和受体复合物以不同的亲和力与IL-4和/或IL-13结合。与IL-4和/或IL-13结合的受体和受体复合物的主要组分为IL-4Rα、IL-13Rα1和IL-13Rα2。这些链作为IL-4Rα/IL-13Rαl(II型IL-4R)或IL-4Rα/γc(I型IL-4R)的单体或异源二聚体表达在细胞表面上。IL-4Rα单体和IL-4Rα/γc异源二聚体与IL-4结合但不与IL-13结合。IL-13Rα1和IL-13Rα2单体与IL-13结合但不与IL-4结合。IL-4Rα/IL-13Rα1异源二聚体既与IL-4结合，也与IL-13结合(Murata等人,Int.J.Hematol.,1999,69,13-20)。Cell surface receptors and receptor complexes bind IL-4 and/or IL-13 with varying affinities. The major components of receptors and receptor complexes that bind IL-4 and/or IL-13 are IL-4Rα, IL-13Rα1 and IL-13Rα2. These chains are expressed on the cell surface as monomers or heterodimers of IL-4Rα/IL-13Rα1 (type II IL-4R) or IL-4Rα/γc (type I IL-4R). IL-4Rα monomer and IL-4Rα/γc heterodimer bind IL-4 but not IL-13. IL-13Rα1 and IL-13Rα2 monomers bind IL-13 but not IL-4. The IL-4Rα/IL-13Rα1 heterodimer binds both IL-4 and IL-13 (Murata et al., Int. J. Hematol., 1999, 69, 13-20).
Th2-型免疫响应促进抗体生成和体液免疫，并被精心设计用来抵御细胞外病原体。Th2细胞是产生Ig的中介体(体液免疫)并产生IL-4、IL-5、IL-6、IL-9、IL-10和IL-13(Tanaka,等人,Cytokine Regulation of Humoral Immunity，251-272,Snapper,编辑，John Wiley和Sons,New York(1996))。Th2-型免疫响应的特征是产生某些类型的细胞因子(如IL-4,IL-13)以及特定类型的抗体(IgE,IgG4)，属于典型的变应性反应，可能造成含泪眼和哮喘症状，如气道炎症和肺中气道肌肉细胞的收缩。Th2-type immune responses promote antibody production and humoral immunity and are carefully designed to defend against extracellular pathogens. Th2 cells are mediators of Ig production (humoral immunity) and produce IL-4, IL-5, IL-6, IL-9, IL-10 and IL-13 (Tanaka, et al., Cytokine Regulation of Humoral Immunity, 251 -272, Snapper, ed., John Wiley and Sons, New York (1996)). Th2-type immune response is characterized by the production of certain types of cytokines (such as IL-4, IL-13) and specific types of antibodies (IgE, IgG4), which is a typical allergic reaction and may cause teary eyes and Asthma symptoms such as airway inflammation and constriction of airway muscle cells in the lungs.
根据其生物学功能，IL-4和IL-13都是具有重要治疗意义的细胞因子，在许多疾病中，包括哮喘，起着关键的作用(Curr Opin Allergy Clin Immunol 2005,Vo.5,161-166)。IL-4已被证明能够抑制自身免疫疾病，IL-4和IL-13都证明具有增强抗肿瘤免疫响应的潜力。由于这两个细胞因子都涉及到变应性疾病的发病机制，因此，这些细胞因子的抑制剂应该能够提供治疗上的益处。According to their biological functions, both IL-4 and IL-13 are therapeutically important cytokines that play key roles in many diseases, including asthma (Curr Opin Allergy Clin Immunol 2005,Vo.5,161-166) . IL-4 has been shown to suppress autoimmune diseases, and both IL-4 and IL-13 have demonstrated the potential to enhance antitumor immune responses. Since both cytokines are involved in the pathogenesis of allergic diseases, inhibitors of these cytokines should provide therapeutic benefit.
相应地，需要能够抑制IL-4、IL-13的改进的活性剂(agent)，以及既能抑制IL-4也能抑制IL-13的单一活性剂。Accordingly, there is a need for improved agents capable of inhibiting IL-4, IL-13, as well as single agents capable of inhibiting both IL-4 and IL-13.
发明概述Summary of the invention
本发明提供可特异结合到IL-4和/或IL-13的人源化单克隆和双特异新颖抗体及其片段和衍生物。有些抗IL-4和/或IL-13单或双特异抗体及其片段可以改变，以防止形成链内二硫键，从而形成在制备和体内使用过程中均稳定的分子。本发明的抗体在此处所述的生物测定中中和IL-4和/或IL-13活性。The present invention provides humanized monoclonal and bispecific novel antibodies and fragments and derivatives thereof that can specifically bind to IL-4 and/or IL-13. Some anti-IL-4 and/or IL-13 mono- or bispecific antibodies and fragments thereof can be altered to prevent the formation of intrachain disulfide bonds, resulting in stable molecules during manufacture and in vivo use. Antibodies of the invention neutralize IL-4 and/or IL-13 activity in the biological assays described herein.
本发明包括所述抗体的可变重链和轻链的氨基酸序列及其对应的核酸序列。The invention includes the amino acid sequences of the variable heavy and light chains of the antibodies and their corresponding nucleic acid sequences.
本发明的另一实施方案包括携带有本发明所述抗体序列的细胞系和载体。Another embodiment of the invention includes cell lines and vectors carrying the antibody sequences of the invention.
本发明的另一实施方案是所述抗体在制备用于治疗与IL-4和/或IL-13功能和代谢有关的疾病和障碍的药物组合物中的用途。尤其是，本发明与癌症、自身免疫缺陷以及由炎症引起或以炎症为特征的疾病，如变应性哮喘和皮炎的治疗有关。Another embodiment of the present invention is the use of said antibodies for the preparation of pharmaceutical compositions for the treatment of diseases and disorders related to IL-4 and/or IL-13 function and metabolism. In particular, the invention is relevant to the treatment of cancer, autoimmune deficiencies, and diseases caused by or characterized by inflammation, such as allergic asthma and dermatitis.
本申请还涉及下述各项：This application also relates to the following:
1.特异结合IL-4的人源化抗体。CLAIMS 1. A humanized antibody specifically binding to IL-4.
2.项1的抗体，其包含由SEQ ID NO:3组成的可变轻链结构域。2. The antibody of item 1, comprising a variable light chain domain consisting of SEQ ID NO:3.
3.项1的抗体，其包含选自SEQ ID NO:4和SEQ ID NO:5的可变重链结构域。3. The antibody of item 1, comprising a variable heavy chain domain selected from SEQ ID NO:4 and SEQ ID NO:5.
4.项1-3中任一项的可选的抗体，其中所述抗体是抗体片段。4. The optional antibody of any one of items 1-3, wherein said antibody is an antibody fragment.
5.项1的抗体，其包含恒定区的CH1、CH2和CH3结构域。5. The antibody according to item 1, which comprises theCH1 ,CH2 andCH3 domains of the constant region.
6.如项5的多肽，其中的IgG抗体是IgG4抗体。6. The polypeptide according to item 5, wherein the IgG antibody is an IgG4 antibody.
7.编码如项1的多肽的核酸。7. A nucleic acid encoding the polypeptide according to item 1.
8.含有如项7的核酸的载体。8. A vector comprising the nucleic acid according to item 7.
9.含有如项8的载体的细胞。9. A cell containing the vector according to item 8.
10.特异结合IL-13的人源化抗体。10. A humanized antibody that specifically binds IL-13.
11.项10的抗体，其包含由SEQ ID NO:1组成的可变轻链结构域。11. The antibody of item 10, comprising a variable light chain domain consisting of SEQ ID NO:1.
12.项11的抗体，其包含由SEQ ID NO:2组成的可变重链结构域。12. The antibody of item 11, comprising a variable heavy chain domain consisting of SEQ ID NO:2.
13.的项10–12中任一项的可选的抗体，其中所述抗体是抗体片段。13. The antibody optional according to any one of items 10-12, wherein said antibody is an antibody fragment.
14.项10-12中的任一项的抗体，其包含恒定区的CH1、CH2和CH3结构域。14. The antibody of any one of items 10-12, comprising theCH1 ,CH2 andCH3 domains of the constant region.
15.如项14的多肽，其中IgG抗体是IgG4抗体。15. The polypeptide according to item 14, wherein the IgG antibody is an IgG4 antibody.
16.编码如项15的多肽的核酸。16. A nucleic acid encoding the polypeptide according to item 15.
17.含有如项16的核酸的载体。17. A vector comprising the nucleic acid according to item 16.
18.含有如项17的载体的细胞。18. A cell containing the vector according to item 17.
19.特异结合IL-13和IL-4的双特异抗体或双特异抗体片段。19. A bispecific antibody or bispecific antibody fragment that specifically binds IL-13 and IL-4.
20.项19的双特异抗体或抗体片段，其进一步包含可变的轻链和可变的重链。20. The diabody or antibody fragment according to item 19, which further comprises a variable light chain and a variable heavy chain.
21.项20的双特异抗体或抗体片段，其中所述可变轻链包含氨基酸序列SEQ IDNO:1和SEQ ID NO:3。21. The bispecific antibody or antibody fragment according to item 20, wherein the variable light chain comprises the amino acid sequences of SEQ ID NO:1 and SEQ ID NO:3.
22.项20的双特异抗体或抗体片段，其中所述可变重链包含氨基酸序列SEQ IDNO:2和SEQ ID NO:5。22. The bispecific antibody or antibody fragment according to item 20, wherein the variable heavy chain comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:5.
23.项20的双特异抗体或抗体片段，其中所述可变重链包含氨基酸序列SEQ IDNO:2和SEQ ID NO:4。23. The bispecific antibody or antibody fragment according to item 20, wherein the variable heavy chain comprises the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:4.
24.项20的双特异抗体或抗体片段，其中所述可变轻链包含氨基酸序列SEQ IDNO:1和SEQ ID NO:3，且所述可变重链包含氨基酸序列SEQ ID NO:2和SEQ ID NO:4。24. The bispecific antibody or antibody fragment of item 20, wherein the variable light chain comprises the amino acid sequences SEQ ID NO:1 and SEQ ID NO:3, and the variable heavy chain comprises the amino acid sequences SEQ ID NO:2 and SEQ ID NO:2 ID NO: 4.
25.项24的双特异抗体或抗体片段，其中所述SEQ ID NO:1和SEQ ID NO:3由肽连接体连接在一起，所述SEQ ID NO:2和SEQ ID NO:4由肽连接体连接在一起。25. The bispecific antibody or antibody fragment of item 24, wherein said SEQ ID NO: 1 and SEQ ID NO: 3 are linked together by a peptide linker, said SEQ ID NO: 2 and SEQ ID NO: 4 are linked by a peptide body connected together.
26.项25的双特异抗体或抗体片段，其中所述肽连接体由SEQ ID NO:6组成。26. The bispecific antibody or antibody fragment according to item 25, wherein the peptide linker consists of SEQ ID NO:6.
27.特异地结合IL-13和IL-13的双特异抗体，其中可变的轻链包含SEQ ID NO:1和SEQ ID NO:3，可变的重链包含SEQ ID NO:2和SEQ ID NO:4，且还包含恒定区结构域。27. The bispecific antibody specifically binding IL-13 and IL-13, wherein the variable light chain comprises SEQ ID NO:1 and SEQ ID NO:3, and the variable heavy chain comprises SEQ ID NO:2 and SEQ ID NO: 4, and also contains the constant region domain.
28.项27的双特异抗体，其中所述恒定区结构域由CH1、CH2、CH3和CL组成。28. The bispecific antibody according to item 27, wherein said constant region domain consists of CH1, CH2, CH3 and CL.
29.包含项1或项8的人源化抗体和可药用载体的药物组合物。29. A pharmaceutical composition comprising the humanized antibody of item 1 or item 8 and a pharmaceutically acceptable carrier.
30.包含在项19–28的可选的双特异抗体或抗体片段和可药用的载体的药物组合物。30. A pharmaceutical composition comprising the optional bispecific antibody or antibody fragment of items 19-28 and a pharmaceutically acceptable carrier.
31.项1或项10的人源化抗体，其进一步与效应分子缀合。31. The humanized antibody of item 1 or item 10, which is further conjugated to an effector molecule.
32.项31的人源化抗体，其中所述效应分子选自：异源多肽、药物、放射性核苷酸和毒素。32. The humanized antibody according to item 31, wherein said effector molecule is selected from the group consisting of heterologous polypeptides, drugs, radionucleotides and toxins.
33.项19–28中任一项的可选的双特异抗体或抗体片段，其进一步与效应分子缀合。33. The optional bispecific antibody or antibody fragment of any one of items 19-28, which is further conjugated to an effector molecule.
34.项33的双特异抗体或片段，其中所述效应分子选自：异源多肽、药物、放射性核苷酸和毒素。34. The bispecific antibody or fragment according to item 33, wherein said effector molecule is selected from the group consisting of heterologous polypeptides, drugs, radionucleotides and toxins.
35.治疗哺乳动物中变应性疾病的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项19-28的可选的双特异抗体或抗体片段。35. A method of treating allergic diseases in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of an optional bispecific antibody or antibody fragment according to items 19-28.
36.治疗哺乳动物中变应性疾病的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项1或10的人源化抗体。36. A method of treating allergic diseases in a mammal, comprising the step of administering a therapeutically effective amount of a humanized antibody according to item 1 or 10 to said mammal.
37.治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项19-28的可选的双特异抗体或抗体片段。37. A method of treating cancer comprising the step of administering to said mammal a therapeutically effective amount of an optional bispecific antibody or antibody fragment according to items 19-28.
38.治疗癌症的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项1或10的人源化抗体。38. A method for treating cancer, comprising the step of administering a therapeutically effective amount of the humanized antibody according to item 1 or 10 to said mammal.
39.治疗哺乳动物中哮喘的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项19-28的可选的双特异抗体或抗体片段。39. A method of treating asthma in a mammal comprising the step of administering to said mammal a therapeutically effective amount of an optional bispecific antibody or antibody fragment according to items 19-28.
40.治疗哺乳动物中哮喘的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项1或10的人源化抗体。40. A method of treating asthma in a mammal, comprising the step of administering a therapeutically effective amount of a humanized antibody according to item 1 or 10 to said mammal.
41.治疗哺乳动物中与IL-4和/或IL-13异常产生有关的疾病之方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项19-28的可选的双特异抗体或抗体片段。41. A method of treating a disease associated with abnormal production of IL-4 and/or IL-13 in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of an optional bisaminoglycan according to items 19-28. Specific antibodies or antibody fragments.
42.治疗哺乳动物中与IL-4和/或IL-13异常产生有关的疾病之方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项1或10的人源化抗体。42. A method for treating a disease associated with abnormal production of IL-4 and/or IL-13 in a mammal, comprising the step of administering a therapeutically effective amount of the humanized antibody according to item 1 or 10 to said mammal .
43.抑制哺乳动物中TH-2介导的反应的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项19-28的可选的双特异抗体或抗体片段。43. A method of inhibiting a TH-2 mediated response in a mammal, comprising the step of administering to said mammal a therapeutically effective amount of an optional bispecific antibody or antibody fragment according to items 19-28.
44.抑制哺乳动物中TH-2介导的反应的方法，其包括下述步骤：向所述哺乳动物施用治疗有效量的根据项1或10的人源化抗体。44. A method of inhibiting a TH-2 mediated response in a mammal, comprising the step of administering a therapeutically effective amount of a humanized antibody according to item 1 or 10 to said mammal.
45.编码如项27的双特异抗体的核酸。45. A nucleic acid encoding the bispecific antibody according to item 27.
46.含有如项45的核酸的载体。46. A vector comprising the nucleic acid according to item 45.
47.含有如项46的载体的细胞。47. A cell containing the vector according to item 46.
本文还叙述了其它特点和优点，将在下列详细说明和附图中阐述。Other features and advantages are described herein and set forth in the following detailed description and accompanying drawings.
附图简述Brief description of the drawings
图1是含有4个多肽链的双特异抗IL-4/IL-13抗体分子的示意图。两个较轻的链由N-VLhB-B13-连接体-VLh8D4-8-CL-C(CL，轻链恒定区)组成，两个较重的链由N-VHhB-B13-连接体-VHh8D4-8-CH1-CH2-CH3-C组成。连接体序列(G4S)2为GGGGSGGGGS(SEQ ID NO:6)。Figure 1 is a schematic diagram of a bispecific anti-IL-4/IL-13 antibody molecule containing 4 polypeptide chains. The two lighter chains consist of N-VLhB-B13 -linker-VLh8D4-8 -CL-C (CL, light chain constant region) and the two heavier chains are connected by N-VHhB-B13 - Body-VH h8D4-8 -CH1-CH2-CH3-C composition. Linker sequence (G4S)2 is GGGGSGGGGS (SEQ ID NO: 6).
图2显示人源化的B-B13抗IL-13抗体(SEQ ID NO:1和2)的可变结构域之氨基酸序列以及人源化的8D4-8抗IL-4抗体(SEQ ID NO:3,4和5)的可变结构域之氨基酸序列。下划线表示对所做的氨基酸改变。粗体表示CDR。Figure 2 shows the amino acid sequence of the variable domains of the humanized B-B13 anti-IL-13 antibody (SEQ ID NO: 1 and 2) and the humanized 8D4-8 anti-IL-4 antibody (SEQ ID NO: Amino acid sequences of the variable domains of 3, 4 and 5). Amino acid changes made to are underlined. Bold indicates CDRs.
本发明之详细说明Detailed Description of the Invention
本发明不局限于本文所述的具体方法学、方案、细胞系、载体或试剂，这是因为它们可发生变动，而不偏离本发明的精神和范围。而且，本文所使用的术语仅出于例示具体实施方案之目的，并非意在限制本发明之范围。除非另有定义，本文所使用的所有技术和科学术语和任何首字母缩略词的含义与本发明领域普通技术人员通常理解的含义相同。实施本发明时可使用任何类似或相当于本文所述的方法和材料，本文仅叙述了例示性方法、设备和材料。The present invention is not limited to particular methodology, protocols, cell lines, vectors or reagents described herein, as these may be varied without departing from the spirit and scope of the invention. Moreover, the terminology used herein is for the purpose of illustrating particular embodiments only and is not intended to limit the scope of the invention. Unless otherwise defined, all technical and scientific terms and any acronyms used herein have the same meaning as commonly understood by one of ordinary skill in the art of the invention. Any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, only exemplary methods, devices and materials are described herein.
出于叙述和披露可能与本发明一同使用和可能在本发明中使用的所报道的蛋白质、酶、载体、宿主细胞和方法学的目的，本文所提及的所有专利和出版物均籍引用之方式整体并入本文。但是，本文任何部分均不可理解为是承认因在先发明的缘故，本发明无权声称早于这些披露的内容。All patents and publications mentioned herein are incorporated by reference for the purpose of describing and disclosing reported proteins, enzymes, vectors, host cells and methodologies that may be used with and in the present invention The method is incorporated into this article as a whole. However, nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.
在教导制备和使用IL-4和/或IL-13相关的方法和目标产物之前，提供了一些术语和短语的下列非限制性定义，以指导技术人员。The following non-limiting definitions of some terms and phrases are provided to guide the skilled artisan before teaching methods and target products related to making and using IL-4 and/or IL-13.
“白介素-4＂(IL-4)涉及天然出现或内源的哺乳动物IL-4蛋白或与天然出现或内源的相应哺乳动物IL-4蛋白具有同样氨基酸序列的蛋白(如重组蛋白，合成蛋白(即用合成有机化学方法制备的蛋白质))。相应地，如此处所定义的，该术语包括成熟的IL-4蛋白、多态或等位变体、IL-4的其它同种型，以及上述的修饰的或未修饰的形式(如脂化(lipidate)或糖基化)。天然出现或内源的IL-4包括野生型蛋白质如成熟的IL-4、多态或等位变体以及其它在哺乳动物(如人，非人灵长类动物)中天然出现的同种型和突变体形式。这些蛋白可从如天然产生IL-4的来源回收或分离出来。这些蛋白质以及与天然出现或内源的相应IL-4有相同氨基酸序列的蛋白质是按相应的哺乳动物之名称命名的。例如，当相应的哺乳动物是人时，该蛋白质则称为人IL-4。本领域已知几种突变IL-4蛋白质，如WO 03/038041中所披露的。"Interleukin-4" (IL-4) refers to a naturally occurring or endogenous mammalian IL-4 protein or a protein having the same amino acid sequence as a naturally occurring or endogenous corresponding mammalian IL-4 protein (e.g. recombinant protein, synthetic Proteins (i.e. proteins prepared by synthetic organic chemistry)). Accordingly, as defined herein, the term includes mature IL-4 proteins, polymorphic or allelic variants, other isoforms of IL-4, and modified or unmodified forms of the above (such as lipidated ( lipidate) or glycosylation). Naturally occurring or endogenous IL-4 includes wild-type proteins such as mature IL-4, polymorphic or allelic variants, and other isoforms that occur naturally in mammals (e.g., humans, non-human primates) and mutant forms. These proteins can be recovered or isolated from sources such as naturally occurring IL-4. These proteins, as well as proteins having the same amino acid sequence as the naturally occurring or endogenous corresponding IL-4, are named after the corresponding mammalian names. For example, when the corresponding mammal is a human, the protein is referred to as human IL-4. Several mutant IL-4 proteins are known in the art, as disclosed in WO 03/038041.
“白介素-13＂(IL-13)指的是天然出现或内源的哺乳动物IL-13蛋白或与天然出现或内源的相应哺乳动物IL-13蛋白具有同样氨基酸序列的蛋白(如重组蛋白，合成蛋白(即用合成有机化学方法制备的蛋白质))。相应地，如此处所定义的，该术语包括成熟的IL-13蛋白、多态或等位变体、IL-13的其它同种型(如通过可选剪接或其它细胞过程所产生的)，以及上述的修饰的或未修饰的形式(如脂化或糖基化)。天然出现或内源的IL-13包括野生型蛋白质如成熟的IL-13、多态或等位变体以及其它在哺乳动物中天然出现的同种型和突变形式(如人，非人灵长类动物)。例如，此处所用的IL-13包括人IL-13变体，其中位于成熟的人IL-13之110位置的Arg被Gin所置换(成熟的IL-13的110位置相当于前体蛋白质的130位置)，Gin与哮喘(特异反应性和非特异反应性哮喘)以及其它IL-13的变体有关。(Heinzmann等人,Hum MoI Genet 9:549-559(2000))。这些蛋白可从如天然产生IL-13的来源回收或分离出来。这些蛋白质以及与天然出现或内源的相应IL-13有相同氨基酸序列的蛋白质是按相应的哺乳动物之名称命名的。例如，当相应的哺乳动物是人时，该蛋白质则称为人IL-13。本领域已知有几种突变IL-13蛋白质，如WO 03/035847中所披露的。"Interleukin-13" (IL-13) refers to a naturally occurring or endogenous mammalian IL-13 protein or a protein having the same amino acid sequence as a naturally occurring or endogenous corresponding mammalian IL-13 protein (e.g. recombinant protein , Synthetic proteins (that is, proteins prepared by synthetic organic chemistry methods)). Accordingly, as defined herein, the term includes mature IL-13 proteins, polymorphic or allelic variants, other isoforms of IL-13 (such as produced by alternative splicing or other cellular processes), and Modified or unmodified forms (eg lipidated or glycosylated) of the above. Naturally occurring or endogenous IL-13 includes wild-type proteins such as mature IL-13, polymorphic or allelic variants, and other isoforms and mutant forms that occur naturally in mammals (e.g., human, non-human primate animals). For example, IL-13 as used herein includes human IL-13 variants in which Arg at position 110 of mature human IL-13 is replaced by Gin (position 110 of mature IL-13 corresponds to position 130 of the precursor protein). position), Gin is associated with asthma (both atopic and non-atopic asthma) and other variants of IL-13. (Heinzmann et al., Hum MoI Genet 9:549-559 (2000)). These proteins can be recovered or isolated from sources such as naturally occurring IL-13. These proteins, as well as proteins having the same amino acid sequence as the naturally occurring or endogenous corresponding IL-13, are named after the corresponding mammalian names. For example, when the corresponding mammal is a human, the protein is referred to as human IL-13. Several mutant IL-13 proteins are known in the art, as disclosed in WO 03/035847.
就抗体链多肽序列而言，短语“基本相同”可理解为表现出与参照多肽序列至少70％、80％、90％、95％或更多的序列同一性的抗体链。就核酸序列而言，该术语可理解为表现出与参照核酸序列至少大于85％、90％、95％或97％或更高的序列同一性的核苷酸序列。With reference to antibody chain polypeptide sequences, the phrase "substantially identical" is understood to mean antibody chains exhibiting at least 70%, 80%, 90%, 95% or more sequence identity to a reference polypeptide sequence. In relation to nucleic acid sequences, the term is understood as meaning a nucleotide sequence exhibiting at least greater than 85%, 90%, 95% or 97% or more sequence identity to a reference nucleic acid sequence.
术语"同一性"或"同源性"可指候选序列中与相应序列残基相同的核苷酸碱基或氨基酸残基的百分数，所述候选序列为与相应序列进行比较，经比对序列和引入空位(若有必要)以实现整段序列的最大同一性百分数且不把任何保守性取代视为序列同一性部分后。无论N端或C端延伸或插入均不应理解为降低同一性或同源性。用于比对的方法和计算机程序均易获得，并为本领域所熟知。可使用序列分析软件测量序列同一性。The terms "identity" or "homology" may refer to the percentage of nucleotide bases or amino acid residues in a candidate sequence that are identical to residues in a corresponding sequence to which the aligned sequences are compared and after introducing gaps (if necessary) to achieve the maximum percent identity over the entire sequence without considering any conservative substitutions as part of the sequence identity. Neither N-terminal or C-terminal extensions or insertions should be understood as reducing identity or homology. Methods and computer programs for alignment are readily available and well known in the art. Sequence identity can be measured using sequence analysis software.
抗体或抗原的"功能片段、变异体、衍生物或类似物"等以及它们的多种形式等短语和术语是指具有与全长目的抗体或抗原在性质上相同的生物活性的化合物或分子。例如，抗IL-4抗体的功能片段或类似物是可结合IL-4分子的片段或类似物，或是可防止或基本上降低配体或激动或拮抗性抗体结合IL-4的能力的片段或类似物。Phrases and terms such as "functional fragments, variants, derivatives, or analogs" of antibodies or antigens, and their various forms refer to compounds or molecules that have the same biological activity as the full-length antibody or antigen of interest. For example, a functional fragment or analog of an anti-IL-4 antibody is a fragment or analog that binds an IL-4 molecule, or that prevents or substantially reduces the ability of a ligand or an agonistic or antagonistic antibody to bind IL-4 or similar.
"取代型"变异体是天然序列中至少一个氨基酸残基被除去并被不同的氨基酸插入其相同位置的变异体。所述取代可为单个的，其中该分子中仅有一个氨基酸被取代；或可为多个的，其中该相同分子有两个或更多的氨基酸被取代。多个取代可位于连续的位点。同样，一个氨基酸可被多个残基取代，其中这样的变异体包括取代和插入二者。"插入型"变异体是一个或多个氨基酸被插入到紧邻一段天然序列某个特定位置处的氨基酸的变异体。紧邻氨基酸意指与该氨基酸的α-羧基或α-氨基官能团连接。"缺失型"变异体是天然氨基酸序列中一个或多个氨基酸被除去的变异体。通常情况下，缺失型变异体在其分子的特定区域内有一个或两个氨基酸被缺失。A "substitution" variant is one in which at least one amino acid residue has been removed from the native sequence and a different amino acid has been inserted at its same position. The substitutions can be single, where only one amino acid is substituted in the molecule, or multiple, where two or more amino acids are substituted in the same molecule. Multiple substitutions may be at consecutive positions. Likewise, an amino acid may be substituted by multiple residues, wherein such variants include both substitutions and insertions. An "insertional" variant is one in which one or more amino acids are inserted immediately adjacent to a specific position in a native sequence. Immediately adjacent to an amino acid means attached to the α-carboxyl or α-amino functional group of the amino acid. A "deletion" variant is one in which one or more amino acids of the native amino acid sequence have been removed. Typically, deletion variants have one or two amino acids deleted from a specific region of the molecule.
术语"抗体"被用于最宽泛的含义，具体涵盖单克隆抗体(包括全长单克隆抗体)、多克隆抗体、多特异性抗体(例如双特异性抗体)、携带一个或多个CDR或源自CDR序列的抗体片段或合成多肽，只要这些多肽表现出所需的生物活性。抗体(Abs)和免疫球蛋白(Igs)是具有相同结构特征的糖蛋白。一般情况下，抗体被认为是具有确定或公认的特异性的Ig。因此，尽管抗体表现出与特异靶的结合特异性，但免疫球蛋白包括抗体和其它缺乏靶特异性的抗体样分子。本发明所述抗体可为任何种类(例如IgG、IgE、IgM、IgD、IgA等)、或亚类(例如IgG1、IgG2、IgG2a、IgG3、IgG4、IgA1、IgA2等)的抗体("类型"和"种类"、以及"亚型"和"亚类"在本文中可互换使用)。天然或野生型(即得自未人工操纵的群体成员)抗体和免疫球蛋白通常为约150,000道尔顿的异四聚体糖蛋白，其由两个相同的轻链(L)和两个相同的重链(H)组成。每条重链的一端具有可变结构域(VH)，随后是多个恒定结构域。每条轻链的一端具有可变结构域(VL)，另一端具有恒定结构域。所谓"未人工操纵"意指未经旨在使其含有或表达外来抗原结合分子的处理。野生型可指一个群体中发现的最普遍的等位基因或种类或指得自未操纵动物的抗体，相比较于等位基因或多态型，或得自以某种形式的操纵例如诱变、使用重组方法等改变该抗原结合分子的氨基酸的变异体或衍生物。The term "antibody" is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (such as bispecific antibodies), Antibody fragments or synthetic polypeptides derived from CDR sequences, as long as these polypeptides exhibit the desired biological activity. Antibodies (Abs) and immunoglobulins (Igs) are glycoproteins that share the same structural characteristics. In general, antibodies are considered to be Igs of defined or recognized specificity. Thus, while antibodies exhibit binding specificity for a specific target, immunoglobulins include antibodies and other antibody-like molecules that lack target specificity. The antibody of the present invention can be of any class (such as IgG, IgE, IgM, IgD, IgA, etc.), or subclass (such as IgG1 , IgG2 , IgG2a , IgG3 , IgG4 , IgA1 , IgA2 , etc.) ("type" and "class", and "subtype" and "subclass" are used interchangeably herein). Native or wild-type (i.e., obtained from members of a population that has not been manipulated) antibodies and immunoglobulins are typically heterotetrameric glycoproteins of about 150,000 Daltons consisting of two identical light chains (L) and two identical heavy chain (H) composition. Each heavy chain has a variable domain (VH ) at one end followed by constant domains. Each light chain has a variable domain (VL ) at one end and a constant domain at the other end. The so-called "not artificially manipulated" means that it has not been treated for the purpose of containing or expressing foreign antigen-binding molecules. Wild type may refer to the most prevalent allele or species found in a population or to antibodies obtained from unmanipulated animals, as compared to alleles or polymorphisms, or from manipulation in some form such as mutagenesis , A variant or derivative in which the amino acid of the antigen-binding molecule is altered by recombinant methods or the like.
正如本文所使用，"抗IL-4抗体"意指可特异结合本文所定义的IL-4的抗体或源自这些抗体的多肽(衍生物)，其包括但不限于抑制或实质降低IL-4与其受体的结合或抑制IL-4活性的分子。As used herein, "anti-IL-4 antibody" means an antibody or a polypeptide (derivative) derived from these antibodies that specifically binds IL-4 as defined herein, including but not limited to inhibiting or substantially reducing IL-4 A molecule that binds to its receptor or inhibits the activity of IL-4.
正如本文所使用，"抗IL-13抗体"意指可特异结合本文所定义的IL-13的抗体或源自这些抗体的多肽(衍生物)，其包括但不限于抑制或实质降低IL-13与其受体的结合或抑制IL-13活性的分子。As used herein, "anti-IL-13 antibody" means an antibody or a polypeptide (derivative) derived from these antibodies that specifically binds IL-13 as defined herein, including but not limited to inhibiting or substantially reducing IL-13 A molecule that binds to its receptor or inhibits the activity of IL-13.
就抗体的可变结构域而言，术语"可变"系指抗体之间有广泛序列差异的相关分子的某些部分，且被用于针对其特异靶的特定抗体的特异识别和结合。但是，可变性在抗体的整个可变结构域内不是均匀分布的。可变性集中在被称为互补决定区域(CDRs；即CDR1、CDR2和CDR3)或超变区的三个区段，它们均位于轻链和重链的可变结构域内。可变结构域内保守程度更高的部分被称为构架(FR)区或构架序列。天然重链和轻链的每个可变结构域均包括四个FR区，其主要采用β-折叠构型，它们籍三个CDRs连接起来，CDRs形成环，所述环连接β-折叠结构并在某些情形下形成部分的β-折叠结构。每条链的CDRs通常被FR区在邻近连接起来，并且借助于来自其它链的CDR，有助于抗体靶结合位点(表位或决定簇)的形成(参看Kabat等人Sequences of Proteins of Immunological Interest,National Instituteof Health,Bethesda,MD(1987))。正如本文所使用，免疫球蛋白氨基酸残基的编号是依据Kabat等人的免疫球蛋白氨基酸残基编号系统而进行的，除非另有说明。一个CDR可具有特异结合关联表位的能力。With respect to the variable domains of antibodies, the term "variable" refers to certain portions of related molecules that differ widely in sequence between antibodies and are used for the specific recognition and binding of a particular antibody against its specific target. However, the variability is not evenly distributed throughout the variable domains of antibodies. The variability is concentrated in three segments called complementarity determining regions (CDRs; namely CDR1, CDR2 and CDR3) or hypervariable regions, which are located within the variable domains of the light and heavy chains. The more conserved portions of variable domains are called the framework (FR) regions or framework sequences. Each variable domain of native heavy and light chains consists of four FR regions, predominantly in a β-sheet configuration, connected by three CDRs that form loops that connect the β-sheet structures and In some cases a partial β-sheet structure is formed. The CDRs of each chain are usually connected adjacently by FR regions and contribute to the formation of the antibody target binding site (epitope or determinant) by means of CDRs from other chains (see Kabat et al. Sequences of Proteins of Immunological Interest, National Institute of Health, Bethesda, MD (1987)). As used herein, numbering of immunoglobulin amino acid residues is according to the immunoglobulin amino acid residue numbering system of Kabat et al., unless otherwise indicated. A CDR may have the ability to specifically bind a cognate epitope.
本发明所用的术语"绞链"或"绞链区"系指包含抗体的第一和第二恒定结构域之间的氨基酸的柔性多肽。The term "hinge" or "hinge region" as used herein refers to a flexible polypeptide comprising the amino acids between the first and second constant domains of an antibody.
术语"抗体片段"系指完整或全长链或抗体的一部分，一般是靶结合区域或可变区域。抗体片段的实例包括但不限于Fab、Fab'、F(ab')2和Fv片段。"功能片段"或"抗IL-4和/或IL-13抗体的类似物"是可防止或实质降低所述受体结合配体或启动信号转导的能力的片段或类似物。正如本文所使用，功能片段一般与"抗体片段"含义相同，且就抗体而论，可指能防止或实质降低所述受体结合配体或启动信号转导的能力的片段，例如Fv、Fab、F(ab')2等等。"Fv"片段由一条重链的可变结构域和一条轻链的可变结构域籍非共价结合方式而形成的二聚体(VH-VL二聚体)组成。在该构型中，每个可变结构域的三个CDRs相互作用，以确定VH-VL二聚体表面上的靶结合位点，与完整抗体的情况一样。所述六个CDRs共同赋予完整抗体的靶结合特异性。但是，即使是单个可变结构域(或仅包括3个靶特异的CDRs的Fv的一半)，仍可具有识别和结合靶的能力。The term "antibody fragment" refers to an entire or full-length chain or portion of an antibody, typically a target binding region or a variable region. Examples of antibody fragments include, but are not limited to, Fab,Fab' , F(ab')2 andFv fragments. A "functional fragment" or "analog of an anti-IL-4 and/or IL-13 antibody" is a fragment or analog that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction. As used herein, a functional fragment generally has the same meaning as "antibody fragment" and, with respect to an antibody, may refer to a fragment that prevents or substantially reduces the ability of the receptor to bind a ligand or initiate signal transduction, such asFv , Fab , F(ab')2 and so on. The "Fv " fragment consists of a dimer (VH -VL dimer) formed by non-covalent association of the variable domain of a heavy chain and the variable domain of a light chain. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of theVH -VL dimer, as is the case with intact antibodies. Together, the six CDRs confer target binding specificity to the intact antibody. However, even a single variable domain (or half of anFv comprising only 3 target-specific CDRs) may still have the ability to recognize and bind a target.
"单链Fv"、"sFv"或"scAb"抗体片段包括抗体的VH和VL结构域，其中这些结构域位于单条多肽链上。一般而言，所述Fv多肽还包括一段位于VH和VL区域之间的多肽连接体，其通常为柔性分子，可使sFv形成适合于靶结合的所需结构。A "single-chain Fv,""sFv ," or "scAb " antibody fragment includes theVH andVL domains of an antibody, wherein these domains are located on a single polypeptide chain. In general, theFv polypeptide also includes a polypeptide linker between theVH andVL regions, which is usually a flexible molecule that allows the sFv to form a desired structure suitable for target binding.
术语"双抗体(diabody)"系指具有两个抗原结合位点的抗体片段，这些片段可包括与同一多肽链的轻链可变结构域(VL)相连的重链可变结构域(VH)。通过使用过短的连接体使得同一条链上的两个可变结构域无法配对，所述双抗体结构域被迫与另一条链的结合结构域配对，生成两个抗原结合位点。The term "diabody" refers to antibody fragments having two antigen-binding sites, which fragments may include a heavy chain variable domain (VL ) linked to a light chain variable domain (V L ) of the same polypeptide chain.H ). By using a linker that is too short to allow pairing of the two variable domains on the same chain, the diabody domain is forced to pair with the binding domain of another chain, creating two antigen-binding sites.
Fab片段含有轻链的可变结构域和恒定结构域以及重链的可变和第一个恒定结构域(CH1)。Fab'片段与Fab片段的不同之处在于前者在CH1结构域的羧基端加入数个残基，以包括一个或多个来自抗体铰链区的半胱氨酸。通过裂解位于F(ab')2胃蛋白酶消化产物的铰链半胱氨酸处的二硫键，可生成Fab'片段。对抗体另外进行酶处理和化学处理可生成其它目的功能片段。TheFab fragment contains the variable and constant domains of the light chain and the variable and first constant domain (CH1 ) of the heavy chain.Fab' fragments differ fromFab fragments by the addition of several residues at the carboxy-terminus of theCH1 domain to include one or more cysteines from the antibody hinge region.Fab' fragments are generated by cleavage of disulfide bonds at the hinge cysteines of F(ab')2 pepsin digests. Additional enzymatic and chemical treatments of antibodies can generate other functional fragments of interest.
术语"线性Fab"系指Miller等所述之四价抗体(Miller等人(2003),JImmunol.170:4854-4861)。"线性Fab"是由一连串相同的CH1-VH结构域组成的，在每一个CH1-VH位置与相同的轻链配对。研发了这些分子是为了增加抗体的效价并透过亲合力效应来增强其功能性亲和力，但是，它们是单特异性的。The term "linear Fab" refers to the tetravalent antibody described by Miller et al. (Miller et al. (2003), J Immunol. 170:4854-4861). A "linear Fab" is composed of a series of identical CH1-VH domains paired with the same light chain at each CH1-VH position. These molecules were developed to increase the titer of antibodies and enhance their functional affinity through avidity effects, however, they are monospecific.
术语"双特异抗体(BsAbs)"系指在同一个分子里组合了两个抗体的抗原结合位点的分子。因此，双特异抗体能够同时结合两个不同的抗原。除了诊断目的之应用之外，双特异抗体通过将强力效应体系转向患病区域，或增加抗体的中和或刺激活性，从而为开发新的治疗方法铺平了道路。The term "bispecific antibodies (BsAbs)" refers to molecules that combine the antigen-binding sites of two antibodies in the same molecule. Thus, bispecific antibodies are capable of binding two different antigens simultaneously. In addition to applications for diagnostic purposes, bispecific antibodies pave the way for the development of new therapeutic approaches by redirecting potent effector systems to diseased areas, or increasing the neutralizing or stimulating activity of antibodies.
最初试图使用化学融合的异缀合物分子将针对不同靶抗原的两个完整抗体的结合特异性偶联(Staerz等人(1985),Nature 314:628-631)。Initial attempts were made to couple the binding specificities of two intact antibodies directed against different target antigens using chemically fused heteroconjugate molecules (Staerz et al. (1985), Nature 314:628-631).
已经透过异杂交瘤技术从杂种杂交瘤中制备了双特异抗体，体外显示其具有与观察到的异缀合物类似的性质(Milstein&Cuello(1983)Nature 305:537-540)。Bispecific antibodies have been prepared from hybrid hybridomas by heterohybridoma technology and shown in vitro to have similar properties to those observed for heteroconjugates (Milstein & Cuello (1983) Nature 305:537-540).
尽管利用如上所述从细胞融合制备的异缀合物或双特异抗体得到了令人有希望的结果，但是几个因素却使它们的大规模治疗应用变得不实际。这些因素包括：体内大异缀合物的快速清除、产生任何一类分子所需要的高劳动强度的技术、需要深入纯化将异缀合物从单缀合物或单特异抗体分离出来，而且通常产率很低。Despite promising results using heteroconjugates or bispecific antibodies prepared from cell fusion as described above, several factors have made their large-scale therapeutic application impractical. These factors include: the rapid clearance of large heteroconjugates in vivo, the labor-intensive techniques required to produce molecules of any class, the need for extensive purification to separate heteroconjugates from monoconjugates or monospecific antibodies, and often The yield is very low.
基因工程越来越多地被用于设计、改变、生产具有一组期望的结合特性和效应物功能的抗体或抗体衍生物。Genetic engineering is increasingly being used to design, alter, and produce antibodies or antibody derivatives with a desired set of binding properties and effector functions.
现已研发了多种重组技术来有效地生产双特异抗体，既可作为抗体片段(Carter等人(1995),J.Hematotherapy 4:463-470；Pluckthun等人(1997)Immunotechology 3:83-105；Todorovska等人(2001)J.Immunol.Methods 248:47-66)，也可作为全长IgG形式(Carter(2001)J.Immunol.Methods 248:7-15)。A variety of recombinant technologies have been developed to efficiently produce bispecific antibodies, both as antibody fragments (Carter et al. (1995), J. Hematotherapy 4:463-470; Pluckthun et al. (1997) Immunotechology 3:83-105 ; Todorovska et al. (2001) J. Immunol. Methods 248:47-66), also available as a full-length IgG format (Carter (2001) J. Immunol. Methods 248:7-15).
将两个不同的scFvs组合产生具有最小分子量的称为sc-BsAbs或Ta-scFvs的双特异抗体形式(Mack等人(1995),Proc.Acad.Sci.USA.92:7021-7025；Mallender等人(1994)J.Biol.Chem.269:199-206)。基因工程上，籍由二聚化官能团如亮氨酸拉链融合两个scFvs从而构建双特异抗体(Kostelny等人(1992)J.Immunol.148:1547-53；de Kruif等人(1996)J.Biol.Chem.271:7630-4)。Combining two different scFvs produces bispecific antibody formats called sc-BsAbs or Ta-scFvs with minimal molecular weight (Mack et al. (1995), Proc. Acad. Sci. USA. 92:7021-7025; Mallender et al. (1994) J. Biol. Chem. 269:199-206). In genetic engineering, bispecific antibodies are constructed by fusing two scFvs with a dimerization functional group such as a leucine zipper (Kostelny et al. (1992) J. Immunol. 148:1547-53; de Kruif et al. (1996) J. Biol. Chem. 271:7630-4).
如上所述，双抗体是很小的二价双特异抗体片段。这些片段在同一肽链上包含通过使用连接体连结到VL的VH，所述连接体太短(少于12个氨基酸)使得同一链上的两个结构域之间无法配对。这些结构域被迫与另一个链上互补的结构域进行分子间配对，从而产生两个抗原结合位点。这些二聚抗体片段，或“双抗体＂，为二价并具有双特异性。(Holliger等人(1993),Proc.Natl.Acad.Sci.USA.90:6444-6448)。双抗体的大小与Fab片段类似。以3到12个氨基酸的连接体连接的VH和VL结构域的多肽链主要形成二聚体(双抗体)，而由0到2个氨基酸残基的连接体连接的则有利于形成三聚体(三抗体(triabody))和四聚体(四抗体(tetrabody))。除了连接体的长度外，寡聚化的确切模式似乎取决于V-结构域的组成和取向(Hudson等人(1999),J Immunol Methods 231:177-189)。双抗体分子最后结构的可预测性很差。As mentioned above, diabodies are small bivalent bispecific antibody fragments. These fragments comprise VH linked to VL on the same peptide chain by using a linker that is too short (less than 12 amino acids) to allow pairing between the two domains on the same chain. These domains are forced into intermolecular pairing with complementary domains on another chain, creating two antigen-binding sites. These dimeric antibody fragments, or "diabodies", are bivalent and bispecific. (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA. 90:6444-6448). Diabodies are similar in size to Fab fragments. Polypeptide chains of the VH and VL domains linked by a linker of 3 to 12 amino acids form predominantly dimers (diabodies), whereas those joined by a linker of 0 to 2 amino acid residues favor trimer formation (triabody) and tetramer (tetrabody). In addition to the length of the linker, the exact pattern of oligomerization appears to depend on the composition and orientation of the V-domain (Hudson et al. (1999), J Immunol Methods 231:177-189). The final structure of the diabody molecule is poorly predictable.
尽管基于sc-BsAbs和双抗体的构建体显示很有趣的临床潜力，但是，已经证明，这样的非共价结合的分子在生理条件下却不够稳定。scFv片段的总体稳定性取决于VL和VH结构域自身的稳定性以及结构域界面的稳定性。已经显示，scFv片段VH-VL界面的稳定性不够是不可逆scFv失活的主要原因，因为肽连接体所允许的界面瞬态打开会使有利于聚集的疏水区块(patch)曝露，因此造成不稳定和低产率(和Plückthun(2001),J.Mol.Biol.305:989-1010)。Although constructs based on sc-BsAbs and diabodies show interesting clinical potential, it has been shown that such non-covalently bound molecules are not sufficiently stable under physiological conditions. The overall stability of scFv fragments depends on the stability of the VL and VH domains themselves and the stability of the domain interfaces. Insufficient stability of the VH-VL interface of scFv fragments has been shown to be a major cause of irreversible scFv inactivation, as the transient opening of the interface permitted by the peptide linker exposes hydrophobic patches that favor aggregation, thus causing undesirable Stable and low yield ( and Plückthun (2001), J. Mol. Biol. 305:989-1010).
US5,989,830中披露了可选的从VH和VL结构域制备双特异二价抗原结合蛋白的方法。这样的双头抗体片段是籍由表达编码两个多肽链的双顺反子载体获得的，其中一个多肽链有两个通过肽连接体串联的VH(VH1-连接体-VH2)，另一个多肽链由互补的VL结构域组成，所述VL结构域由肽连接体串联连接(VL1-连接体-VL2)。US5,989,830描述，每个连接体应该包含至少10个氨基酸残基。An alternative method for making bispecific bivalent antigen binding proteins from VH and VL domains is disclosed in US 5,989,830. Such double-headed antibody fragments are obtained by expressing a bicistronic vector encoding two polypeptide chains, one of which has two VHs connected in series via a peptide linker (VH1-linker-VH2), The chain consists of complementary VL domains connected in tandem by a peptide linker (VL1-linker-VL2). US 5,989,830 describes that each linker should contain at least 10 amino acid residues.
US2005/0003403 A1描述了价态增加的多价蛋白复合物(PPC)。多价蛋白复合物包含两个多肽链，它们通常彼此侧向排列。每个多肽链通常包含3或4个包含氨基酸序列的“v区＂，当与相对的多肽链上的对应的v区匹配时，这些氨基酸序列能够形成抗原结合位点。每个多肽链上可用多达大约6个“v区＂。每个多肽链上的v区彼此线性连接，可由散开的连接区连接。当排列成PPC形式时，每个多肽链上的v区形成单个的抗原结合位点。复合物可能包含一种或几种结合特异性。US2005/0003403 A1 describes polyvalent protein complexes (PPCs) with increased valence states. A multivalent protein complex consists of two polypeptide chains, which are usually arranged laterally to each other. Each polypeptide chain typically contains 3 or 4 "v-regions" comprising amino acid sequences that, when matched with corresponding v-regions on the opposing polypeptide chain, are capable of forming an antigen binding site. Up to about 6 "v regions" may be used on each polypeptide chain. The V regions on each polypeptide chain are linked to each other linearly, and may be connected by interspersed linking regions. When arranged in a PPC format, the v-regions on each polypeptide chain form a single antigen-binding site. Complexes may contain one or several binding specificities.
但是，利用这样的分子显示了聚集，不稳定性以及很差的表达产率(Wu等人(2001)Prot.Eng.14:1025-1033)。这些是表达基于单链的抗体时可能出现的典型稳定性问题。(和Plückthun(2001),J.Mol.Biol.305:989-1010)。However, aggregation, instability and poor expression yields have been shown with such molecules (Wu et al. (2001) Prot. Eng. 14:1025-1033). These are typical stability issues that can arise when expressing single chain-based antibodies. ( and Plückthun (2001), J. Mol. Biol. 305:989-1010).
因此，本发明的目的就是要提供双特异多价抗体，利用其可避免形成聚集物。此外，它应具有稳定性，使之可用于治疗应用。Therefore, the object of the present invention is to provide bispecific multivalent antibodies, by which the formation of aggregates can be avoided. Furthermore, it should be stable so that it can be used for therapeutic applications.
本文所使用的术语"单克隆抗体"系指得自基本同源抗体群的抗体，即组成该群体的单个抗体除可能有少数存在天然发生的突变外，均是相同的。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, ie, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations.
本文的单克隆抗体具体包括"嵌合"抗体，其中一部分重链和/或轻链与源自某特定物种或属于某特定抗体种类或亚类(类型或亚型)的相应序列相同或同源，其中所述链的其余部分与源自另一物种或属于另一抗体种类或亚类的抗体以及所述抗体片段的相应序列相同或同源，只要这些抗体片段表现出结合IL-4和/或IL-13或影响IL-4和/或IL-13活性或代谢的所需生物活性(US专利号4,816,567；和Morrison等人,Proc Natl Acad Sci USA81:6851(1984))。因此，来自同一类别抗体的CDR可移植到不同种类或亚类抗体的FR。Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chains are identical or homologous to corresponding sequences derived from a particular species or belonging to a particular antibody class or subclass (type or subtype) , wherein the remainder of said chain is identical or homologous to the corresponding sequences of antibodies derived from another species or belonging to another antibody class or subclass, as well as said antibody fragments, so long as these antibody fragments exhibit binding to IL-4 and/or or IL-13 or a desired biological activity that affects IL-4 and/or IL-13 activity or metabolism (US Patent No. 4,816,567; and Morrison et al., Proc Natl Acad Sci USA 81:6851 (1984)). Thus, CDRs from antibodies of the same class can be grafted to FRs of antibodies of a different class or subclass.
单克隆抗体是高度特异性的抗体，针对单个靶位点、表位或决定簇。而且，与通常包括针对抗原不同决定簇(表位)的不同抗体的常规(多克隆)抗体制品不同的是，每个单克隆抗体针对靶上的单个决定簇。除其特异性以外，单克隆抗体在宿主细胞内的合成是有利的，不受其它免疫球蛋白的污染，并为克隆提供了编码其抗体链的相关基因和mRNA。修饰语"单克隆"提示得自基本同源抗体群的抗体的性质，其不应理解为需要籍任何特定方法产生所述抗体。例如，用于本发明的单克隆抗体可从噬菌体抗体库经使用众所周知的技术分离或从多克隆制品纯化。依据本发明使用的亲代单克隆抗体可通过Kohler等人在Nature256:495(1975)中所述的杂交瘤方法制备，或通过本领域内众所周知的重组方法制备。Monoclonal antibodies are highly specific antibodies directed against a single target site, epitope or determinant. Furthermore, each monoclonal antibody is directed against a single determinant on the target, unlike conventional (polyclonal) antibody preparations, which generally comprise different antibodies directed against different determinants (epitopes) of the antigen. In addition to their specificity, the synthesis of monoclonal antibodies in host cells is advantageous, free from contamination by other immunoglobulins, and provides cloning with the relevant genes and mRNA encoding their antibody chains. The modifier "monoclonal" suggests the nature of an antibody obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies useful in the present invention can be isolated from phage antibody libraries or purified from polyclonal preparations using well known techniques. Parental monoclonal antibodies used in accordance with the invention can be prepared by the hybridoma method described by Kohler et al., Nature 256:495 (1975), or by recombinant methods well known in the art.
本发明所使用的术语"多价抗体"系指包含两个或多个抗原结合位点、因而能够结合两个或多个具有相同或不同结构的抗原之抗体。术语"二价"表示抗体包含两个抗原结合位点。术语"四价"表示抗体包含四个抗原结合位点。The term "multivalent antibody" used in the present invention refers to an antibody that contains two or more antigen-combining sites and thus is capable of binding two or more antigens with the same or different structures. The term "bivalent" means that the antibody comprises two antigen combining sites. The term "tetravalent" means that the antibody contains four antigen combining sites.
本发明所使用的术语"抗原结合位点"系指抗体中这样的部份，其包含特异地与抗原之部份或全部结合，并与抗原之部份或全部成互补的区域。当抗原很大时，抗体可能只能结合到抗原的一个特定的部份，该部份被称为表位。抗原结合结构域可由一个或多个抗体可变结构域提供。优选的是，抗原结合结构域是由抗体轻链可变结构域(VL)和抗体重链可变结构域(VH)结合形成的。The term "antigen-combining site" used in the present invention refers to such a part of an antibody, which includes a region that specifically binds to a part or all of an antigen and is complementary to a part or all of an antigen. When an antigen is large, antibodies may only bind to a specific part of the antigen, called an epitope. The antigen binding domain may be provided by one or more antibody variable domains. Preferably, the antigen binding domain is formed by the combination of an antibody light chain variable domain (VL) and an antibody heavy chain variable domain (VH).
本发明所使用的术语"抗原"系指能够被本发明的抗体所结合的分子或分子之一部份。抗原可有一个或多于一个表位。本发明抗体可识别的抗原之实例包括但不限于血清蛋白如细胞因子，例如IL-4、IL5、IL9和IL-13，生物活性肽、细胞表面分子如受体、转运蛋白、离子通道、病毒和细菌蛋白。The term "antigen" used in the present invention refers to a molecule or a part of a molecule capable of being bound by the antibody of the present invention. An antigen may have one or more than one epitope. Examples of antigens that can be recognized by the antibodies of the present invention include, but are not limited to, serum proteins such as cytokines, such as IL-4, IL5, IL9 and IL-13, biologically active peptides, cell surface molecules such as receptors, transporters, ion channels, viruses and bacterial proteins.
本发明所使用的术语"单特异的"系指本发明的多价抗体仅可识别出一个抗原，所有的抗原结合位点都是相同的。The term "monospecific" used in the present invention means that the multivalent antibody of the present invention can only recognize one antigen, and all antigen-binding sites are the same.
本发明所使用的术语"双特异的"系指本发明的多价抗体可在同一或两个不同的抗原上识别出两个不同的表位。The term "bispecific" used in the present invention means that the multivalent antibody of the present invention can recognize two different epitopes on the same or two different antigens.
本发明所使用的术语"多特异的"系指本发明的多价抗体可在同一或多个不同的抗原上识别出多个不同的表位。The term "multispecific" used in the present invention means that the multivalent antibody of the present invention can recognize multiple different epitopes on the same or multiple different antigens.
本发明所使用的术语"连接体"系指一个经过调整连接本发明抗体结构可变结构域的肽。肽连接体可包含任何氨基酸，优选的为甘氨酸(G)和丝氨酸(S)。在重链多肽和轻链多肽之间或内部，连接体彼此可为相同或不同。此外，连接体的长度可为1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19或20个氨基酸。重链结构域和轻链结构域的优选的肽连接体单元为GGGGS。重链和轻链的连接体单元的数目可以是相同的(对称的顺序)，也可以是彼此不同的(非对称的顺序)。The term "linker" used in the present invention refers to a peptide adjusted to link the variable domains of the antibody structure of the present invention. The peptide linker may comprise any amino acid, preferably glycine (G) and serine (S). Between or within the heavy chain polypeptide and the light chain polypeptide, the linkers may be the same or different from each other. Furthermore, the linker can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids in length. A preferred peptide linker unit for heavy and light chain domains is GGGGS. The number of linker units of the heavy and light chains may be the same (symmetrical order) or different from each other (asymmetrical order).
肽连接体优选足够长以便能够提供足够的柔性程度以防止抗体部分例如通过位阻干扰彼此的活性，从而允许蛋白质适当地折叠，如有必要，还可让抗体分子与同一细胞上两个或多个可能分得很开的受体相互作用；然而，优选的是足够短以允许抗体部份在细胞中保持稳定。The peptide linker is preferably long enough to provide a sufficient degree of flexibility to prevent the antibody moieties from interfering with each other's activity, e.g. The receptor interactions may be spaced apart; however, it is preferably short enough to allow the antibody moiety to remain stable in the cell.
因此，肽连接体的长度、组成和/或构像可很容易地由本领域技术人员选定，以便优化多价抗体所需要的性质。Thus, the length, composition and/or conformation of the peptide linker can be readily selected by one skilled in the art in order to optimize the desired properties of the multivalent antibody.
非人(例如鼠)抗体的"人源化"形式是嵌合的免疫球蛋白、免疫球蛋白链或它们的片段(例如抗体的Fv、Fab、Fab'、F(ab')2或其它靶结合亚序列)，与人抗体相比，其含有源自非人免疫球蛋白的序列。一般而言，所述人源化抗体基本上总共包括一个、典型为两个的可变结构域，其中所有或基本上所有CDR区域对应于非人免疫球蛋白的CDR区域，以及所有或基本上所有FR区域是人免疫球蛋白模板序列的FR区域。所述人源化抗体还可包括至少一部分免疫球蛋白恒定区(Fc)，通常是所选择的人免疫球蛋白模板的恒定区。一般而言，目的是拥有在人体内免疫原性最低的抗体分子。因此，有可能一个或多个CDRs中的一个或多个氨基酸也可被更换成在人宿主体内免疫原性更低的氨基酸，却基本不最小化所述一个或多个CDRs对IL-4和/或IL-13的特异结合功能。或者，FR可为非人的，但免疫原性最强的那些氨基酸被替换为免疫原性较低的氨基酸。但是，上文所讨论的CDR移植并非是获得人源化抗体的唯一途径。例如，仅修饰CDR区域可能不够充分，因为构架区残基参与决定CDR环的三维结构和抗体与其配体的总亲和力的现象并非鲜见。因此，可实施任何方法，以使非人亲代抗体分子被修饰为对人免疫原性更低的抗体分子，而且并非总是需要与人抗体的全局序列同一性(global sequence identity)。因此，也可通过例如仅取代数个残基实现人源化，尤其是取代暴露在抗体分子上且没有被包埋在该分子内部因此不易接近宿主免疫系统的残基。本文在取代抗体分子上的"活动"或"柔性"残基方面教导了该方法，其目标是降低或减缓所形成分子的免疫原性，却不破坏所述抗体对其表位或决定簇的特异性。例如参见Studnicka等人,Prot Eng 7(6)805-814,1994；Mol Imm 44:1986-1988,2007；Sims等人,J Immunol151:2296(1993)；Chothia等人,J Mol Biol 196:901(1987)；Carter等人,Proc Natl AcadSci USA 89:4285(1992)；Presta等人,J Immunol 151:2623(1993),WO 2006/042333和美国专利号5,869,619。"Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (e.g., Fv,Fab ,Fab' , F(ab')2of antibodies or other target binding subsequences), which contain sequences derived from non-human immunoglobulins, as compared to human antibodies. In general, the humanized antibody will comprise substantially a total of one, typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin, and all or substantially All FR regions are FR regions of human immunoglobulin template sequences. The humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc ), typically that of a human immunoglobulin template of choice. In general, the aim is to have antibody molecules that are the least immunogenic in humans. Thus, it is possible that one or more amino acids in one or more CDRs may also be replaced with an amino acid that is less immunogenic in a human host without substantially minimizing the effect of said one or more CDRs on IL-4 and /or the specific binding function of IL-13. Alternatively, the FRs may be non-human, but with those amino acids that are most immunogenic replaced by less immunogenic amino acids. However, the CDR grafting discussed above is not the only way to obtain humanized antibodies. For example, modifying CDR regions alone may not be sufficient, as it is not uncommon for framework region residues to participate in determining the three-dimensional structure of CDR loops and the overall affinity of an antibody for its ligand. Thus, any method can be implemented to modify the non-human parent antibody molecule to be less immunogenic to humans, and global sequence identity to human antibodies is not always required. Humanization can thus also be achieved, for example, by substituting only a few residues, especially those residues that are exposed on the antibody molecule and are not buried inside the molecule and therefore less accessible to the host's immune system. The method is taught herein in terms of substituting "mobile" or "flexible" residues on an antibody molecule with the goal of reducing or slowing the immunogenicity of the resulting molecule without disrupting the antibody's affinity for its epitope or determinant. specificity. See, eg, Studnicka et al., Prot Eng 7(6) 805-814, 1994; Mol Imm 44:1986-1988, 2007; Sims et al., J Immunol 151:2296 (1993); Chothia et al., J Mol Biol 196:901 (1987); Carter et al., Proc Natl AcadSci USA 89:4285 (1992); Presta et al., J Immunol 151:2623 (1993), WO 2006/042333 and US Patent No. 5,869,619.
感兴趣的人源化方法基于抗体的分子柔性在免疫识别期间和其时的影响。蛋白质柔性与蛋白分子的分子运动有关。蛋白质柔性是整个蛋白、部分蛋白或单个氨基酸残基采取构象集合(ensemble)的能力，其中所述构象各自显著不同。有关蛋白质柔性的信息可通过进行蛋白质X射线结晶学实验(参见例如Kundu等人,2002,Biophys J 83:723–732.)、核磁共振实验(参见例如Freedberg等人,J Am Chem Soc 1998,120(31):7916-7923)或进行分子动力学(MD)模拟而获得。在计算机上进行蛋白质的MD模拟，可通过计算原子之间的物理相互作用而允许测定所有蛋白质原子在一段时间内的运动。MD模拟的输出是所研究蛋白在模拟时间段内的轨迹。该轨迹是蛋白质构象的集合，也称为快照(snapshot)，所述蛋白质构象在模拟期间例如每1皮秒(ps)周期性采样。正是通过分析快照的集合，人们能够定量蛋白质氨基酸残基的柔性。因此，就含有柔性残基的多肽而言，柔性残基是采取不同构象集合的残基。MD方法为本领域所周知，参见例如Brooks等人,Proteins:A TheoreticalPerspective of Dynamics,Structure and Thermodynamics(Wiley,New York,1988)。数种软件可进行MD模拟，例如Amber(参见Case等人(2005)J Comp Chem 26:1668-1688),Charmm(参见Brooks等人(1983)J Comp Chem 4:187-217；和MacKerell等人(1998)TheEncyclopedia of Computational Chemistry vol.1:271-177,Schleyer等人eds.Chichester:John Wiley&Sons)或Impact(参见Rizzo等人,J Am Chem Soc；2000；122(51):12898-12900)。Interesting humanization methods are based on the influence of the molecular flexibility of antibodies during and upon immune recognition. Protein flexibility is related to the molecular motion of protein molecules. Protein flexibility is the ability of an entire protein, part of a protein, or individual amino acid residues to adopt an ensemble of conformations where the conformations are each significantly different. Information about protein flexibility can be obtained by performing protein X-ray crystallography experiments (see for example Kundu et al., 2002, Biophys J 83:723-732.), nuclear magnetic resonance experiments (see for example Freedberg et al., J Am Chem Soc 1998, 120 (31):7916-7923) or obtained by molecular dynamics (MD) simulations. Performing MD simulations of proteins on a computer allows the determination of the motion of all protein atoms over time by calculating the physical interactions between atoms. The output of an MD simulation is the trajectory of the protein under study over the simulated time period. The trajectory is a collection, also known as a snapshot, of protein conformations that are sampled periodically during the simulation, for example every 1 picosecond (ps). It is by analyzing collections of snapshots that one is able to quantify the flexibility of protein amino acid residues. Thus, for a polypeptide containing flexible residues, a flexible residue is one that adopts a collection of different conformations. MD methods are well known in the art, see eg Brooks et al., Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics (Wiley, New York, 1988). Several software programs can perform MD simulations, such as Amber (see Case et al. (2005) J Comp Chem 26:1668-1688), Charmm (see Brooks et al. (1983) J Comp Chem 4:187-217; and MacKerell et al. (1998) The Encyclopedia of Computational Chemistry vol.1:271-177, Schleyer et al. eds. Chichester: John Wiley & Sons) or Impact (see Rizzo et al., J Am Chem Soc; 2000; 122(51):12898-12900).
大多数蛋白质复合物享有相对较大和较平的被包埋表面，业已表明结合伴侣蛋白的柔性提供了其弹性的来源，使得它们能够构象性彼此适应(Structure(2000)8,R137-R142)。因此，业已表明"诱导契合"的实例在蛋白质-蛋白质的界面中起了主要作用。此外，稳定增长的数据体表明蛋白质实际上结合多种形状、大小和组成的配体(Protein Science(2002)11:184-187)，且构象多样性似乎是识别不同伴侣蛋白能力的关键组分(Science(2003)299,1362-1367)。柔性残基参与了蛋白质-蛋白质伴侣蛋白的结合(Structure(2006)14,683-693)。Most protein complexes share a relatively large and flat embedded surface, and it has been shown that the flexibility of binding partners provides a source of their elasticity, enabling them to adapt conformationally to each other (Structure (2000) 8, R137-R142). Thus, instances of "induced fit" have been shown to play a major role at protein-protein interfaces. Furthermore, a steadily growing body of data indicates that proteins actually bind ligands of diverse shapes, sizes and compositions (Protein Science (2002) 11:184-187), and that conformational diversity appears to be a key component of the ability to recognize different chaperones (Science (2003) 299, 1362-1367). Flexible residues are involved in protein-protein chaperone binding (Structure (2006) 14, 683-693).
柔性残基可采取许多构象，它们提供了相互作用区域的集合，其有可能被记忆B细胞识别并触发免疫原性反应。因此，可通过修饰许多来自构架区的残基使抗体人源化，使得构象集合和受修饰抗体所展示的识别区域集合尽可能类似于人抗体所采取的构象集合和识别区域集合。Flexible residues can adopt many conformations that provide a collection of interacting regions that have the potential to be recognized by memory B cells and trigger an immunogenic response. Thus, antibodies can be humanized by modifying a number of residues from the framework regions such that the set of conformations and set of recognition regions displayed by the modified antibody resembles as closely as possible those adopted by human antibodies.
这一点可通过以下列方法修饰有限数目的残基得以实现：(1)构建亲代mAb的同源模型，进行MD模拟；(2)分析柔性残基和鉴定非人抗体分子最柔性的残基，以及鉴定有可能是异质性或降解反应来源的残基或基序；(3)鉴定展示出与亲代抗体最相似的识别区域集合的人抗体；(4)测定待突变的柔性残基，可能为异质性和降解来源的残基或基序也发生突变；(5)检查是否存在已知T细胞或B细胞表位。使用本文所教导的MD计算经使用隐式溶剂模型可发现柔性残基，该溶剂模型描述了水溶剂与蛋白质原子在模拟期间内的相互作用的原因。一旦在可变轻链和重链内鉴定出一组柔性残基，即鉴定出与目的抗体高度类似的一组人重链和轻链可变区域构架。例如可使用BLAST搜索在抗体人种系序列的数据库中在一组柔性残基上进行。还可通过比较亲代mAb的动力学与种系规范结构库的动力学来进行。检索时排除CDR残基和邻近残基，以确保对于抗原的高度亲和力得以保留。This can be achieved by modifying a limited number of residues by: (1) constructing a homology model of the parental mAb and performing MD simulations; (2) analyzing flexible residues and identifying the most flexible residues in nonhuman antibody molecules, and identify residues or motifs that are likely sources of heterogeneity or degradation responses; (3) identify human antibodies that display the set of recognition regions most similar to the parental antibody; (4) determine flexible residues to mutate, possibly Residues or motifs that are sources of heterogeneity and degradation are also mutated; (5) Check for the presence of known T-cell or B-cell epitopes. Using the MD calculations taught herein flexible residues can be found using an implicit solvent model that describes the reason for the interaction of the aqueous solvent with the protein atoms during the simulation. Once a set of flexible residues within the variable light and heavy chains has been identified, a set of human heavy and light chain variable region frameworks that are highly similar to the antibody of interest is identified. For example, a BLAST search can be performed on a database of antibody human germline sequences on a flexible set of residues. It can also be done by comparing the kinetics of the parental mAbs to the kinetics of a repertoire of germline canonical structures. CDR residues and neighboring residues were excluded from the search to ensure that the high affinity for the antigen was preserved.
随后取代柔性残基。当几个人残基表现出类似同源性时，还通过可能影响人源化抗体的溶液行为的残基性质驱动该选择。例如，暴露的柔性环上优选极性残基，而不是疏水残基。为不稳定性和异质性潜在来源的残基也发生突变，即使它们在CDRs上被发现。这些残基包括暴露的甲硫氨酸，因为亚砜形成可能是由于氧基、对酸敏感的键例如Asp-Pro二肽的键的蛋白水解切割(Drug Dev Res(2004)61:137-154)；位于暴露的天冬酰胺残基和随后的小氨基酸例如Gly、Ser、Ala、His、Asn或Cys处的脱酰胺作用位点(J Chromatog(2006)837:35-43)和N-糖基化位点例如Asn-X-Ser/Thr位点。一般情况下，暴露的甲硫氨酸会被Leu所取代，暴露的天冬酰胺会被谷氨酰胺或天冬氨酸所取代，或其随后的残基会被更换。对于糖基化位点(Asn-X-Ser/Thr)而言，可更换Asn或Ser/Thr残基。Subsequent substitution of flexible residues. This selection was also driven by the nature of the residues which may affect the solution behavior of the humanized antibody when several human residues exhibited similar homology. For example, polar residues are preferred over hydrophobic residues on exposed flexible loops. Residues that are potential sources of instability and heterogeneity were also mutated, even if they were found on CDRs. These residues include exposed methionines, as sulfoxide formation may be due to proteolytic cleavage of oxy, acid-sensitive linkages such as those of the Asp-Pro dipeptide (Drug Dev Res (2004) 61:137-154 ); deamidation sites at exposed asparagine residues and subsequent small amino acids such as Gly, Ser, Ala, His, Asn or Cys (J Chromatog (2006) 837:35-43) and N-glycans Kylation sites such as Asn-X-Ser/Thr sites. Typically, exposed methionines are replaced by Leu, exposed asparagine by glutamine or aspartic acid, or subsequent residues are replaced. For glycosylation sites (Asn-X-Ser/Thr), the Asn or Ser/Thr residues can be substituted.
检查形成的复合序列是否存在已知的B细胞或线性T细胞表位。例如，可利用公众可用的IEDB进行检索。如果在该复合序列中发现已知表位，则检索和取代另一组人序列。The resulting composite sequence is checked for known B-cell or linear T-cell epitopes. For example, searching may be performed using the publicly available IEDB. If known epitopes are found in this composite sequence, another set of human sequences is retrieved and substituted.
与US专利号5,639,641的表面重塑方法不同的是，该方法探讨了B细胞介导和T细胞介导的免疫原性反应。该方法还避免了有时使用CDR移植可观察到的活性丢失问题(US专利号5,530,101)。此外，在设计和选择过程中还考虑了稳定性和溶解度的问题，从而产生对于低免疫原性、高抗原亲和力和改善生物物理性质最优化的抗体。Unlike the resurfacing method of US Patent No. 5,639,641, this method explores both B cell-mediated and T cell-mediated immunogenic responses. This approach also avoids the problem of loss of activity sometimes observed with CDR grafting (US Patent No. 5,530,101). In addition, issues of stability and solubility were considered during the design and selection process, resulting in antibodies optimized for low immunogenicity, high antigen affinity, and improved biophysical properties.
例如US专利号5,639,641披露了用于表面重塑抗体的策略和方法以及降低抗体在不同宿主体内免疫原性的其它方法。简而言之，在一优选方法中，(1)生成抗体重链和轻链可变区域库的位置比对，以产生重链和轻链可变区域构架表面暴露的位置，其中所有可变区域的比对位置至少约98％是相同的；(2)定义一组重链和轻链可变区域构架表面暴露的氨基酸残基，以用于非人例如啮齿类动物抗体(或其抗体片段)；(3)鉴定一组与所述啮齿类动物表面暴露的氨基酸残基最为一致的重链和轻链可变区域构架表面暴露的氨基酸残基；以及(4)除了在所述啮齿类动物抗体CDR的任何残基的任何原子以内的氨基酸残基外，将步骤(2)所确定的一组重链和轻链可变区域构架表面的暴露氨基酸替换为步骤(3)所鉴定的一组重链和轻链可变区域构架表面的暴露氨基酸残基，以生成保留结合特异性的人源化例如啮齿类动物抗体。For example, US Patent No. 5,639,641 discloses strategies and methods for resurfacing antibodies and other methods of reducing the immunogenicity of antibodies in different hosts. Briefly, in a preferred method, (1) generate a positional alignment of antibody heavy and light chain variable region repertoires to generate heavy and light chain variable region framework surface exposed positions in which all variable The aligned positions of the regions are at least about 98% identical; (2) defining a set of surface-exposed amino acid residues of the heavy and light chain variable domain frameworks for use in non-human, e.g., rodent antibodies (or antibody fragments thereof) ); (3) identifying a set of surface-exposed amino acid residues that are most consistent with the surface-exposed amino acid residues in the rodent; Any atom of any residue of an antibody CDR Outside the amino acid residues within, the exposed amino acids on the surface of a set of heavy chain and light chain variable domain frameworks determined in step (2) are replaced by a set of heavy chain and light chain variable domain frameworks identified in step (3) Surface exposed amino acid residues to generate humanized eg rodent antibodies that retain binding specificity.
抗体可利用许多其它技术包括CDR移植(EPO 0 239 400；WO 91/09967和US专利号5,530,101以及5,585,089)、贴面(veneering)或表面重塑(EPO 0 592 106；EPO 0 519596；Padlan,1991,Molec Imm 28(4/5):489-498；Studnicka等人,1994,Prot Eng 7(6):805-814和Roguska等人,1994,PNAS 91:969-973)和链改组(US专利号5,565,332)进行人源化。人抗体可籍许多本领域知晓的方法进行制备，包括但不限于噬菌体展示方法(参见US专利号4,444,887,4,716,111,5,545,806和5,814,318和WO 98/46645,WO 98/50433,WO 98/24893,WO 98/16654,WO 96/34096,WO 96/33735和WO 91/10741)，使用转基因动物例如啮齿类动物，使用嵌合细胞等。Antibodies can utilize a number of other techniques including CDR grafting (EPO 0 239 400; WO 91/09967 and US Patent Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EPO 0 592 106; EPO 0 519596; , Molec Imm 28(4/5):489-498; Studnicka et al., 1994, Prot Eng 7(6):805-814 and Roguska et al., 1994, PNAS 91:969-973) and chain shuffling (US Patent No. 5,565,332) for humanization. Human antibodies can be prepared by a number of methods known in the art, including but not limited to phage display methods (see US Pat. /16654, WO 96/34096, WO 96/33735 and WO 91/10741), using transgenic animals such as rodents, using chimeric cells and the like.
"抗体同源物"或“同源物”系指任何按此处所教导特异结合IL-4和/或IL-13的分子。因此，抗体同源物包括天然或重组抗体、不论修饰与否，保留了目的生物学性质(例如结合IL-4或IL-13)的抗体部分，例如Fab或Fv分子、单链抗体以及携带一个或多个CDR区域的多肽等。所述同源物的氨基酸序列无需与天然存在的抗体氨基酸序列相同，但可经改变或修饰以携带取代氨基酸、插入氨基酸、缺失氨基酸、除蛋白质上正常存在的20个氨基酸以外的氨基酸等，以获得具有增强或其它有利性质的多肽。"Antibody homologue" or "homologue" refers to any molecule that specifically binds IL-4 and/or IL-13 as taught herein. Thus, antibody homologues include natural or recombinant antibodies, portions of antibodies, whether modified or not, that retain biological properties of interest (e.g., binding IL-4 or IL-13), such asFab orFv molecules, single chain antibodies, and Polypeptides carrying one or more CDR regions, etc. The amino acid sequence of such homologues need not be identical to the amino acid sequence of a naturally occurring antibody, but may be altered or modified to carry substituted amino acids, inserted amino acids, deleted amino acids, amino acids other than the 20 amino acids normally present on the protein, etc., to carry Polypeptides having enhanced or other advantageous properties are obtained.
具有同源序列的抗体是其氨基酸序列与本发明的IL-4,IL-13或双特异IL-4/IL-13抗体的氨基酸序列具有序列同源性的抗体。优选地，同源性位于本发明抗体的可变区域的氨基酸序列。应用于本文氨基酸序列的"序列同源性"定义为经例如根据Pearson&Lipman,Proc Natl Acad Sci USA 85,2444-2448(1988)的FASTA检索方法测定与其它氨基酸序列具有至少约90％、91％、92％、93％、94％或更多的序列同源性，更优选地为至少约95％、96％、97％、98％或99％的序列同源性。The antibody with a homologous sequence is an antibody whose amino acid sequence has sequence homology with the amino acid sequence of the IL-4, IL-13 or bispecific IL-4/IL-13 antibody of the present invention. Preferably, the homology is in the amino acid sequences of the variable regions of the antibodies of the invention. "Sequence homology" as applied to amino acid sequences herein is defined as having at least about 90%, 91%, 92%, 93%, 94% or more sequence identity, more preferably at least about 95%, 96%, 97%, 98% or 99% sequence identity.
嵌合抗体是具有不同抗体部分的抗体，其源自不同来源例如不同抗体、不同种类的抗体、不同动物物种，例如具有源自鼠单克隆抗体的可变区与人免疫球蛋白恒定区配对的抗体等。因此，人源化抗体是一种嵌合抗体。用于生产嵌合抗体的方法为本领域所知晓，参见例如Morrison,1985,Science 229:1202；Oi等人,1986,BioTechniques 4:214；Gillies等人,1989,J Immunol Methods 125:191-202和US专利号5,807,715,4,816,567和4,816,397。A chimeric antibody is an antibody that has different antibody portions derived from a different source such as a different antibody, a different class of antibody, a different animal species, for example having variable regions derived from a murine monoclonal antibody paired with human immunoglobulin constant regions. Antibodies, etc. Thus, a humanized antibody is a chimeric antibody. Methods for producing chimeric antibodies are known in the art, see e.g. Morrison, 1985, Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al., 1989, J Immunol Methods 125:191-202 and US Patent Nos. 5,807,715, 4,816,567 and 4,816,397.
人工抗体包括scFv片段、嵌合抗体、双抗体、三抗体、四抗体和mru(参见Winter&Milstein的综述,1991,Nature 349:293-299；和Hudson,1999,Curr Opin Imm 11:548-557)，其中每种均具有抗原结合或表位结合能力。在单链Fv片段(scFv)中，抗体的VH和VL结构域籍柔性胜肽连接起来。一般情况下，所述连接体是约为15个氨基酸的肽。如果连接体小得多，例如为5个氨基酸，则会形成双抗体。抗体的最小结合单元为CDR，通常为具有足够特异识别和结合能力的重链CDR2。这种片段被称为分子识别单元或mru。几个该类mrus可籍短的连接体肽连接到一起，从而形成具有高于单个mru亲合力的人工结合蛋白。Artificial antibodies include scFv fragments, chimeric antibodies, diabodies, triabodies, tetrabodies, and mru (see review by Winter & Milstein, 1991, Nature 349:293-299; and Hudson, 1999, Curr Opin Imm 11:548-557), Each of these has antigen-binding or epitope-binding ability. In single-chainFv fragments (scFv ), theVH andVL domains of an antibody are linked by a flexible peptide. Typically, the linker is a peptide of about 15 amino acids. If the linker is much smaller, eg 5 amino acids, a diabody will form. The smallest binding unit of an antibody is CDR, usually heavy chain CDR2 with sufficient specific recognition and binding ability. Such fragments are known as molecular recognition units, or MRUs. Several such MRUs can be linked together via short linker peptides to form artificial binding proteins with higher affinities than a single MRU.
目的抗体的功能等效物也包括在本发明的范围以内。术语"功能等效物"包括带有同源序列的抗体、抗体同源物、嵌合抗体、人工抗体和修饰的抗体，例如，其中每个功能等效物是按照其结合IL-4和/或IL-13、抑制IL-4和/或IL-13信号传导能力或功能，或抑制IL-4和/或IL-13结合到其受体的能力定义的。技术人员会理解名为"抗体片段"的一组分子与名为"功能等效物"的一组有重迭。制备保留IL-4和/或IL-13结合能力的功能等效物的方法为本领域技术人员知晓，并为例如WO 93/21319,EPO Ser.No.239,400,WO 89/09622,EPOSer.No.338,745和EPO Ser.No.332,424所披露。Functional equivalents of the antibody of interest are also included within the scope of the present invention. The term "functional equivalent" includes antibodies with homologous sequences, antibody homologues, chimeric antibodies, artificial antibodies and modified antibodies, for example, wherein each functional equivalent is based on its binding to IL-4 and/or or IL-13, the ability or function to inhibit IL-4 and/or IL-13 signaling, or the ability to inhibit binding of IL-4 and/or IL-13 to its receptor. The skilled person will understand that the group of molecules named "antibody fragments" overlaps with the group named "functional equivalents". Methods for preparing functional equivalents that retain IL-4 and/or IL-13 binding ability are known to those skilled in the art and are described, for example, in WO 93/21319, EPO Ser.No.239,400, WO 89/09622, EPOSer.No. .338,745 and EPO Ser. No. 332,424.
本申请的功能等效物还包括修饰抗体，例如被任何类型的分子经共价连接至抗体而修饰的抗体。例如，修饰抗体包括业已被已知的保护/阻断基经过例如糖基化、乙酰化、聚乙二醇化、脱酰胺化、磷酸化、酰胺化、衍生化，蛋白水解切割、连接到细胞配体、连接到毒素或细胞毒性部分或其它蛋白等修饰的抗体。共价连接无需这样的抗体，所述抗体免疫于生成抗个体基因型反应。所述修饰可通过已知技术实现，其包括但不限于特定化学切割、乙酰化、甲酰化、代谢合成等。此外，被修饰抗体可含有一个或多个非典型的氨基酸。Functional equivalents of the present application also include modified antibodies, for example antibodies modified by any type of molecule covalently attached to the antibody. For example, modified antibodies include known protecting/blocking groups such as glycosylation, acetylation, pegylation, deamidation, phosphorylation, amidation, derivatization, proteolytic cleavage, attachment to cellular ligands, etc. Modified antibodies such as bodies, attached to toxins or cytotoxic moieties, or other proteins. Covalent attachment does not require antibodies immune to the generation of anti-idiotypic responses. The modification can be achieved by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis and the like. In addition, a modified antibody may contain one or more atypical amino acids.
本领域技术人员可利用许多技术来实现结合亲和力的优化。一般情况下，这些技术包括取代目的位点处的多种氨基酸残基、随后筛选分析突变多肽对关联抗原或表位的结合亲和力。Optimization of binding affinity can be achieved by a number of techniques available to those skilled in the art. Generally, these techniques involve substituting multiple amino acid residues at the site of interest, followed by screening to analyze the binding affinity of the mutant polypeptide to the cognate antigen or epitope.
一旦抗体被鉴定和分离，通常可用于生成变异抗体或突变体或突变蛋白质，其中例如所述抗体的一个或多个高变区的一个或多个氨基酸残基被改变。可选地或额外地，可向抗体引入构架区残基的一个或多个变动(例如取代)，其中这些变动导致抗体突变体对IL-4和/或IL-13的结合亲和力升高。可经修饰的构架区残基的实例包括非共价直接结合抗原的残基(Amit等人,Science 233:747-753(1986))；影响CDR构象或与其相互作用的残基(Chothia等人,J Mol Biol 196:901-917(1987))；和/或参与VL-VH界面的残基(EP 239400)。在某些实施方案中，一个或多个这类构架区残基的修饰导致抗体与关联抗原的结合亲和力增加。例如，在本发明的该实施方案中可改变约1个到约5个构架区残基。有时，这足以产生适合用于临床前试验的抗体突变体，即使其中高变区的任何残基都没有改变。但正常情况下抗体突变体可包括一个或多个高变区变动。也可改变恒定区以获得理想或更加理想的效应物性质。Once an antibody has been identified and isolated, it can generally be used to generate variant antibodies or mutants or muteins in which, for example, one or more amino acid residues of one or more hypervariable regions of the antibody are altered. Alternatively or additionally, one or more alterations (eg, substitutions) of framework region residues may be introduced into the antibody, wherein these alterations result in increased binding affinity of the antibody mutant for IL-4 and/or IL-13. Examples of framework region residues that may be modified include residues that directly bind antigen non-covalently (Amit et al., Science 233:747-753 (1986)); residues that affect or interact with CDR conformation (Chothia et al. , J Mol Biol 196:901-917 (1987)); and/or residues involved in theVL -VH interface (EP 239400). In certain embodiments, modification of one or more such framework region residues results in increased binding affinity of the antibody for the cognate antigen. For example, from about 1 to about 5 framework region residues may be altered in this embodiment of the invention. Sometimes, this is sufficient to generate antibody mutants suitable for preclinical testing, even in which no residues in the hypervariable regions are altered. Normally, however, antibody mutants may include one or more hypervariable region changes. The constant regions can also be altered to achieve desired or more desirable effector properties.
被变动的高变区残基可随机改变，尤其是当亲代抗体的初始结合亲和力可使随机产生的抗体突变体容易在本文所教导的测定中就改变的结合进行筛选。The hypervariable region residues that are altered can be altered randomly, especially when the initial binding affinity of the parent antibody allows for easy screening of randomly generated antibody mutants for altered binding in the assays taught herein.
一种获得抗体突变体例如CDR突变体的方法是"丙氨酸扫描诱变"(Cunningham&Wells,Science 244:1081-1085(1989)和Cunningham&Wells,Proc Nat Acad Sci USA 84:6434-6437(1991))。一个或多个高变区残基被丙氨酸或聚丙氨酸残基替换。对所述取代表现出功能敏感性的这些高变区残基随后通过在或就取代位点引入进一步或其它突变而精修。因此，尽管事先确定了引入氨基酸序列变动的位点，但突变本身的性质无需事先确定。根据被扫描残基的所需性质，可尝试使用其它氨基酸进行类似取代。One method of obtaining antibody mutants, such as CDR mutants, is "alanine scanning mutagenesis" (Cunningham & Wells, Science 244:1081-1085 (1989) and Cunningham & Wells, Proc Nat Acad Sci USA 84:6434-6437 (1991)) . One or more hypervariable region residues are replaced by alanine or polyalanine residues. Those hypervariable region residues exhibiting functional sensitivity to the substitutions are subsequently refined by introducing further or other mutations at or with respect to the sites of substitution. Thus, although the sites to introduce amino acid sequence changes are identified in advance, the nature of the mutation itself need not be determined in advance. Depending on the desired properties of the residue being scanned, similar substitutions with other amino acids can be attempted.
鉴定待修饰氨基酸残基更为系统的方法包括鉴定参与结合IL-4和/或IL-13的高变区残基和很少或没有参与IL-4和/或IL-13结合的高变区残基。进行非结合高变区残基的丙氨酸扫描，其中检测每个ala突变体对IL-4和/或IL-13的结合的增强。在另一实施方案中，选择明显参与IL-4和/或IL-13结合的这些残基进行修饰。修饰可包括缺失残基或在目的残基邻近插入一个或多个残基。但是，正常情况下修饰包括以另一个氨基酸取代残基。保守性取代可以是第一取代。如果该取代导致生物活性发生变化(例如结合亲和力)，那么可进行另一次保守取代以确定是否得到更多实质性变化。A more systematic approach to identifying amino acid residues to modify involves identifying hypervariable region residues involved in binding IL-4 and/or IL-13 and hypervariable region residues involved in little or no IL-4 and/or IL-13 binding Residues. An alanine scan of non-binding hypervariable region residues was performed in which each ala mutant was tested for enhanced binding to IL-4 and/or IL-13. In another embodiment, those residues that are significantly involved in IL-4 and/or IL-13 binding are selected for modification. Modifications may include deletion of residues or insertion of one or more residues adjacent to a residue of interest. Normally, however, modifications include substitution of a residue with another amino acid. A conservative substitution can be a first substitution. If the substitution results in a change in biological activity (eg, binding affinity), then another conservative substitution can be made to determine if a more substantial change results.
通过选择其性质与正常情况下位于某位点处的氨基酸有更多实质差异的氨基酸，可对抗体范围和生物性质的呈现进行更为实质性的修饰。因此，在维持下列情况下可进行该取代：(a)取代区域的多肽骨架结构，例如片层或螺旋构象；(b)该分子在靶位点处的电荷或疏水性，或(c)侧链的大小。By selecting amino acids whose properties differ more substantially from those normally located at a position, more substantial modifications can be made to the range and presentation of biological properties of the antibody. Thus, the substitution can be made while maintaining: (a) the polypeptide backbone structure of the substituted region, such as a sheet or helical conformation; (b) the charge or hydrophobicity of the molecule at the target site, or (c) the side chain size.
例如，根据常见侧链性质，天然存在的氨基酸可分为以下几组：For example, naturally occurring amino acids can be divided into the following groups based on common side chain properties:
(1)疏水：甲硫氨酸(M或met)、丙氨酸(A或ala)、缬氨酸(V或val)、亮氨酸(L或leu)和异亮氨酸(I或ile)；(1) Hydrophobic: methionine (M or met), alanine (A or ala), valine (V or val), leucine (L or leu) and isoleucine (I or ile );
(2)中性、亲水：半胱氨酸(C或cys)、丝氨酸(S或ser)、苏氨酸(T或thr)、天冬酰胺(N或asn)和谷氨酰胺(Q或gln)；(2) Neutral and hydrophilic: cysteine (C or cys), serine (S or ser), threonine (T or thr), asparagine (N or asn) and glutamine (Q or gln);
(3)酸性：天冬氨酸(D或asp)和谷氨酸(E或glu)；(3) Acidity: aspartic acid (D or asp) and glutamic acid (E or glu);
(4)碱性：组氨酸(H或his)、赖氨酸(K或lys)和精氨酸(R或arg)；(4) Basic: histidine (H or his), lysine (K or lys) and arginine (R or arg);
(5)影响链取向的残基：甘氨酸(G或gly)和脯氨酸(P或pro)，以及(5) Residues affecting chain orientation: glycine (G or gly) and proline (P or pro), and
(6)芳香：色氨酸(W或trp)、酪氨酸(Y或tyr)苯丙氨酸(F或phe)。(6) Aroma: tryptophan (W or trp), tyrosine (Y or tyr) phenylalanine (F or phe).
非保守取代需要以来自另一组的氨基酸调换氨基酸。保守性取代需要将一氨基酸交换成组内另一氨基酸。Non-conservative substitutions entail exchanging an amino acid with an amino acid from another group. Conservative substitutions entail exchanging one amino acid for another within a group.
优选的氨基酸取代包括下列氨基酸取代，其：(1)降低对蛋白水解的敏感性，(2)降低对氧化的敏感性，(3)改变结合亲和力以及(4)赋予或修饰这些类似物的其它生理-化学或功能性质。类似物可包括除天然存在肽序列以外的序列的多种突变蛋白质。例如，可对天然存在的序列(优选在形成分子间接触的区域以外的多肽部分)进行单个或多个氨基酸的取代(优选为保守性氨基酸取代)。除R基或侧链的大小或构象的改变以外，保守性氨基酸取代不应实质性改变亲代序列的结构特征(例如氨基酸替换不应倾向于打断亲代序列中存在的螺旋，或破坏表征亲代序列的其它类型的二级结构(Proteins,Structures andMolecular Principles,Creighton,编辑，W.H.Freeman and Company,New York(1984))；Introduction to Protein Structure(Branden&Tooze,eds.,Garland Publishing,NewYork,N.Y.(1991))；和Thornton等人Nature 354:105(1991)。Preferred amino acid substitutions include those that: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity, and (4) confer or modify other properties of these analogs. Physiological-chemical or functional properties. Analogs may include various muteins of sequences other than the naturally occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made to naturally occurring sequences (preferably portions of the polypeptide other than regions where intermolecular contacts are formed). Conservative amino acid substitutions should not substantially alter the structural features of the parental sequence other than changes in the size or conformation of the R group or side chain (e.g. amino acid substitutions should not tend to break helices present in the parental sequence, or disrupt the characteristic Other types of secondary structures (Proteins, Structures and Molecular Principles, Creighton, ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden & Tooze, eds., Garland Publishing, New York, N.Y. (1991)) and Thornton et al. Nature 354:105 (1991).
通常情况下，生物学性质改善的抗体突变体具有与亲代抗人IL-4和/或IL-13抗体的重链或轻链可变结构域的氨基酸序列至少75％的氨基酸序列同一性或相似性，至少80％、至少85％、至少90％以及通常为至少95％的同一性的氨基酸序列。就亲代抗体序列而言，同一性或相似性在本文中定义为经比对序列和引入空位(若有必要)以实现最大的百分比序列同一性后，候选序列的与亲代抗体残基相同(即相同残基)或类似(即基于相同侧链性质而来自同一组的氨基酸残基，参见上文)的氨基酸残基百分比。Typically, antibody mutants with improved biological properties have at least 75% amino acid sequence identity or similarity to the amino acid sequence of the heavy or light chain variable domains of the parental anti-human IL-4 and/or IL-13 antibody Amino acid sequences that are at least 80%, at least 85%, at least 90%, and usually at least 95% identical. With respect to parental antibody sequences, identity or similarity is defined herein as the residues of the candidate sequence that are identical (i.e. identical residues) or similar (ie amino acid residues from the same group based on the same side chain properties, see above).
或者，通过重链和轻链的FR和CDR区域或抗IL-4、抗IL-13或双特异IL-4/IL-13抗体的Fc区域的系统突变，可生成抗体突变体。生成抗体突变体的另一种方法包括使用噬菌体展示进行亲和力成熟(Hawkins等人,J Mol Biol 254:889-896(1992)和Lowman等人,Biochemistry 30(45):10832-10838(1991))。已知噬菌体外壳蛋白融合(Smith,Science228:1315(1985)；Scott&Smith,Science 249:386(1990)；Cwirla等人,Proc Natl AcadSci USA 8:309(1990)；Devlin等人,Science 249:404(1990)；Wells&Lowman,Curr OpinStruct Biol 2:597(1992)和US5,223,409)可用于将所展示的蛋白或肽的表型与编码它们的噬菌体颗粒的基因型联系在一起。抗体的Fab结构域也已被展示在噬菌体上(McCafferty等人,Nature 348:552(1990)；Barbas等人,Proc Natl Acad Sci USA 88:7978(1991)；和Garrard等人,Biotechnol 9:1373(1991))。Alternatively, antibody mutants can be generated by systematic mutation of the FR and CDR regions of the heavy and light chains or theFc region of anti-IL-4, anti-IL-13 or bispecific IL-4/IL-13 antibodies. Another method for generating antibody mutants involves affinity maturation using phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838 (1991)) . Phage coat protein fusions are known (Smith, Science 228:1315 (1985); Scott & Smith, Science 249:386 (1990); Cwirla et al, Proc Natl AcadSci USA 8:309 (1990); Devlin et al, Science 249:404 ( 1990); Wells & Lowman, Curr OpinStruct Biol 2:597 (1992) and US 5,223,409) can be used to correlate the phenotype of displayed proteins or peptides with the genotype of the phage particles encoding them. TheFab domains of antibodies have also been displayed on phage (McCafferty et al., Nature 348:552 (1990); Barbas et al., Proc Natl Acad Sci USA 88:7978 (1991); and Garrard et al., Biotechnol 9: 1373 (1991)).
单价噬菌体展示由下述组成：将一组蛋白质变体作为噬菌体外壳蛋白融合体展示于噬菌体颗粒上(Bass等人,Proteins 8:309(1990)。以前业已通过连续应用诱变、单价噬菌体展示和功能分析(Lowman&Wells,J Mol Biol 234:564 578(1993)；US专利号5,534,617)如通过集中于抗体的CDR区域(Barbas等人,Proc Natl Acad Sci USA 91:3809(1994)；和Yang等人,JMol Biol 254:392(1995))实现了亲和力成熟或多种蛋白平衡结合亲和力的提高。Monovalent phage display consists of displaying a panel of protein variants on phage particles as fusions to the phage coat protein (Bass et al., Proteins 8:309 (1990). Functional analysis (Lowman & Wells, J Mol Biol 234:564 578 (1993); US Pat. No. 5,534,617) such as by focusing on the CDR regions of the antibody (Barbas et al., Proc Natl Acad Sci USA 91:3809 (1994); and Yang et al. , JMol Biol 254:392 (1995)) achieved affinity maturation or the improvement of the equilibrium binding affinity of multiple proteins.
可在噬菌体颗粒上构建许多(例如106或更多)其序列特定位置上有差异的蛋白变异体的库，其中每个噬菌体颗粒含有编码特定蛋白变异体的DNA。经使用固定化抗原进行多轮亲和力纯化后，分离单个噬菌体克隆，并从DNA推演出所展示蛋白的氨基酸序列。Libraries of many (eg,106 or more) protein variants that differ at specific positions in their sequence can be constructed on phage particles, where each phage particle contains DNA encoding a particular protein variant. After multiple rounds of affinity purification using immobilized antigen, individual phage clones are isolated and the amino acid sequence of the displayed protein is deduced from the DNA.
产生抗体突变体后，可依据本文教导测定该分子相对于亲代抗体的生物活性。正如上文指出，这涉及测定抗体的结合亲和力和/或其它生物活性或物理性质。在本发明的一个优选实施方案中，制备一组抗体突变体，筛选其对抗原的结合亲和力。任选地可对选自筛选的一个或多个抗体突变体进行一次或多次另外的生物活性测定，以确定抗体突变体具有新的或改进的性质。在一个优选实施方案中，所述抗体突变体保留了以亲代抗体类似或更好/更高的结合亲和力结合IL-4和/或IL-13的能力。After antibody mutants are generated, the biological activity of the molecule relative to the parent antibody can be determined according to the teachings herein. As noted above, this involves determining the binding affinity and/or other biological activity or physical properties of the antibody. In a preferred embodiment of the invention, a panel of antibody mutants is prepared and screened for their binding affinity to the antigen. One or more additional biological activity assays can optionally be performed on one or more antibody mutants selected from the screen to determine that the antibody mutants have new or improved properties. In a preferred embodiment, said antibody mutant retains the ability to bind IL-4 and/or IL-13 with a similar or better/higher binding affinity of the parental antibody.
通常根据抗体想要的用途，可对依此选择的抗体突变体进行进一步修饰。这类修饰可包括进一步改变氨基酸序列，与异源多肽的融合和/或共价修饰。例如，一般可使用丝氨酸取代未参与维持抗体突变体适宜构象的半胱氨酸残基，以改善该分子的氧化稳定性和防止异常交联。相反，可向抗体加入半胱氨酸以提高稳定性(尤其当抗体是抗体片段例如Fv片段时)。Antibody mutants thus selected may be further modified, generally depending on the intended use of the antibody. Such modifications may include further changes in amino acid sequence, fusions to heterologous polypeptides and/or covalent modifications. For example, cysteine residues not involved in maintaining the appropriate conformation of the antibody mutant can generally be substituted with serine to improve the oxidative stability of the molecule and to prevent aberrant crosslinking. Conversely, cysteines can be added to antibodies to increase stability (especially when the antibody is an antibody fragment such as anFv fragment).
另一类型的抗体突变体具有改变的糖基化模式。可通过缺失抗体上发现的一个或多个糖类部分和/或加入一个或多个抗体上没有的糖基化位点实现这一点。抗体的糖基化一般为N连接到Asn或O连接到Ser或Thr。三肽序列，天冬酰胺-X-丝氨酸和天冬酰胺-X-苏氨酸是糖类部分经酶催化连接到天冬酰胺侧链的常见识别序列，其中X是除脯氨酸以外的任何氨基酸。例如N-乙酰半乳糖胺、半乳糖、岩藻糖或木糖连接到羟基氨基酸，大多数为丝氨酸或苏氨酸，尽管也可使用5-羟脯氨酸或5-羟赖氨酸。向原抗体的序列加入或取代一个或多个丝氨酸或苏氨酸残基可增加O-连接糖基化的概率。Another type of antibody mutant has an altered glycosylation pattern. This can be achieved by deleting one or more carbohydrate moieties found on the antibody and/or adding one or more glycosylation sites not found on the antibody. Glycosylation of antibodies is generally N-linked to Asn or O-linked to Ser or Thr. The tripeptide sequences, asparagine-X-serine and asparagine-X-threonine, are common recognition sequences for the enzymatic attachment of carbohydrate moieties to the asparagine side chain, where X is any amino acid. For example N-acetylgalactosamine, galactose, fucose or xylose are linked to a hydroxyamino acid, mostly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Addition or substitution of one or more serine or threonine residues to the sequence of the primary antibody can increase the probability of O-linked glycosylation.
理想情况下可修饰本发明所述抗体的效应物功能，以增强抗体的效用。例如，可在Fc区引入半胱氨酸残基，从而在该区域形成链间二硫键。依此生成的同型二聚抗体可具有改善的内化能力和/或增加的补体-介导的细胞杀伤以及抗体依赖的细胞毒性(ADCC)，参见Caron等人,J Exp Med 176:1191-1195(1992)和Shopes,Immunol 148:2918-2922(1993)。或者，可改造具有两个Fc区的抗体，因而该抗体可具有增强的补体裂解和ADCC能力，参见Stevenson等人,Anti-Cancer Drug Design 3:219 230(1989)。Desirably, the effector functions of the antibodies of the invention can be modified to enhance the utility of the antibodies. For example, cysteine residues can be introduced in theFc region to form interchain disulfide bonds in this region. Homodimeric antibodies thus generated may have improved internalization capabilities and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC), see Caron et al., J Exp Med 176:1191-1195 (1992) and Shopes, Immunol 148:2918-2922 (1993). Alternatively, antibodies can be engineered to have twoFc regions and thus have enhanced complement lysis and ADCC capabilities, see Stevenson et al., Anti-Cancer Drug Design 3:219 230 (1989).
抗体的共价修饰涵盖在本发明的范围内。可通过化学合成或通过抗体的酶切割或化学切割(若可行)实现这一点。通过抗体的靶向氨基酸残基与有机衍生剂发生反应可向抗体分子引入抗体的其它类型共价修饰，其中有机衍生剂能够与所选侧链或与N端或C端残基反应。Covalent modification of antibodies is encompassed within the scope of the invention. This can be achieved by chemical synthesis or by enzymatic or chemical cleavage of the antibody, as applicable. Other types of covalent modifications of antibodies can be introduced into the antibody molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or with N- or C-terminal residues.
半胱氨酰残基可与α-卤代乙酸酯(以及相应的胺)例如氯乙酸或氯乙酰胺发生反应，生成羧甲基或羧酰胺甲基(carboxyamidomethyl)衍生物。还可通过例如与溴三氟丙酮、α-溴-β-(5-咪唑基)丙酸、氯乙酰磷酸盐、N-烷基马来酰亚胺、3-硝基-2-吡啶二硫化物、甲基2-吡啶二硫化物、p-chloromercuribenzoate、2-chloromercura-4-硝基苯酚或氯-7-硝基苯并-2-氧杂-1,3-二唑发生反应，衍化得到半胱氨酰残基。Cysteinyl residues can be reacted with α-haloacetates (and corresponding amines) such as chloroacetic acid or chloroacetamide to give carboxymethyl or carboxyamidomethyl derivatives. It can also be obtained by, for example, reacting with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridine disulfide compound, methyl 2-pyridine disulfide, p-chloromercurabenzoate, 2-chloromercura-4-nitrophenol or chloro-7-nitrobenzo-2-oxa-1,3-oxadiazole react to obtain Cysteinyl residues.
可通过与焦碳酸二乙酯在pH 5.5-7.0下反应衍化得到组氨酰残基。还可使用对溴苯酰甲基溴，该反应优选在pH 6.0的0.1M二甲胂酸钠中进行。Histidyl residues can be derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0. p-Bromophenacyl bromide can also be used and the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
赖氨酰(lysinyl)和α末端残基可与琥珀酸或其它羧酸酐发生反应，以逆转残基的电荷。用于衍化得到含有α-氨基残基的其它适宜试剂包括亚氨酸酯例如甲基picolinimidate、磷酸吡哆醛、吡哆醛、氯硼氢化物、三硝基苯磺酸、O-甲基异脲和2,4-戊二酮，氨基酸可用乙醛酸被转氨酶催化。Lysinyl and alpha terminal residues can be reacted with succinic acid or other carboxylic anhydrides to reverse the charge of the residues. Other suitable reagents for derivatization to α-amino containing residues include imidates such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methyliso Urea and 2,4-pentanedione, amino acids can be catalyzed by transaminases with glyoxylate.
可通过与一种或多种常规试剂例如苯甲酰甲醛、2,3-丁二酮、1,2-环己二酮和茚三酮反应来修饰精氨酰残基。精氨酸残基的衍化通常需要碱性反应条件。而且，这些试剂可与赖氨酸以及精氨酸的ε-氨基发生反应。Arginyl residues can be modified by reaction with one or more conventional reagents such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues generally requires basic reaction conditions. Furthermore, these reagents can react with lysine as well as the epsilon-amino group of arginine.
可使用芳香族重氮化合物或四硝基甲烷进行酪氨酰残基的特异修饰。例如，使用N-乙酰基咪唑和四硝基甲烷分别形成O-乙酰酪氨酰种类和3-硝基衍生物。使用125I或131I可碘化酪氨酰残基以制备用于放射免疫测定的标记蛋白。Specific modification of tyrosyl residues can be performed using aromatic diazonium compounds or tetranitromethane. For example, N-acetylimidazole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be iodinated using125 I or131 I to prepare labeled proteins for radioimmunoassays.
通过与碳二亚胺(R-N＝C＝C-R')反应，羧基侧链基团(天冬酰基或谷氨酰基)可被修饰，其中R和R'可为不同的烷基，例如1-环己基-3-(2-吗啉基-4-乙基)碳二亚胺或1-乙基-3-(4-氮鎓-4,4-二甲戊基)碳二亚胺。而且，天冬氨酰和谷氨酰残基经与铵离子反应可被转化为天冬酰胺酰基和谷氨酰胺酰基残基。The carboxyl side chain group (asparagyl or glutamyl) can be modified by reaction with carbodiimide (R-N=C=C-R'), where R and R' can be different alkyl groups, such as 1 - cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonium-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues can be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
谷氨酰胺酰基和天冬酰胺酰基残基常在中性或碱性条件下被脱去酰氨基，分别生成相应的谷氨酰和天冬酰残基。这些残基脱去酰胺的形式落在本发明的范围以内。Glutaminyl and asparaginyl residues are often deamidated under neutral or alkaline conditions to yield the corresponding glutamyl and asparaginyl residues, respectively. The deamidated form of these residues is within the scope of the invention.
其它修饰包括脯氨酸和赖氨酸的羟化、丝氨酰或苏氨酰残基的羟基磷酸化、赖氨酸、精氨酸和组氨酸侧链α-氨基的甲基化(Creighton,Proteins:Structure andMolecular Properties,W.H.Freeman&Co.,San Francisco,pp.79-86(1983))、N端胺的乙酰化和任意C端羧基的酰胺化。Other modifications include hydroxylation of proline and lysine, hydroxyl phosphorylation of seryl or threonyl residues, methylation of α-amino groups of lysine, arginine and histidine side chains (Creighton , Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.79-86 (1983)), acetylation of N-terminal amine and amidation of any C-terminal carboxyl group.
另一类型的共价修饰涉及通过化学方式或酶方式将糖苷偶联到抗体上。这些方法不需要在宿主细胞内产生具有N-连接或O-连接的糖基化能力的抗体。根据所使用的偶联方法，糖可被连接到：(a)精氨酸和组氨酸；(b)游离羧基；(c)游离硫氢基，例如半胱氨酸的硫氢基；(d)游离羟基，例如丝氨酸、苏氨酸或羟脯氨酸的羟基；(e)芳香残基例如苯丙氨酸、酪氨酸或色氨酸的芳香残基；或(f)谷氨酰胺的酰氨。WO 87/05330和Aplin&Wriston,CRCCrit Rev Biochem,pp.259-306(1981)叙述了这类方法。Another type of covalent modification involves chemical or enzymatic coupling of glycosides to the antibody. These methods do not require the production of antibodies capable of N-linked or O-linked glycosylation in host cells. Depending on the coupling method used, sugars can be attached to: (a) arginine and histidine; (b) free carboxyl groups; (c) free sulfhydryl groups, such as that of cysteine; ( d) free hydroxyl groups such as those of serine, threonine or hydroxyproline; (e) aromatic residues such as those of phenylalanine, tyrosine or tryptophan; or (f) glutamine of amide. Such methods are described in WO 87/05330 and Aplin & Wriston, CRCCrit Rev Biochem, pp. 259-306 (1981).
可通过化学或酶方式除去抗体上的任何糖类部分。例如化学去糖基化需要抗体暴露于化合物三氟甲磺酸或相当的化合物，从而导致除连接糖(N-乙酰基葡糖胺或N-乙酰基半乳糖胺)以外的大多数或全部糖的切割，同时保持抗体的完整性。例如Hakimuddin等人,Arch Biochem Biophys 259:52(1987)和Edge等人,Anal Biochem 118:131(1981)叙述了化学去糖基化。可通过例如Thotakura等人,Meth Enzymol 138:350(1987)所述的许多内切糖苷酶和外切糖苷酶中的任意实现抗体上糖类部分的酶切割。Any carbohydrate moieties on the antibody can be removed chemically or enzymatically. For example chemical deglycosylation requires exposure of the antibody to the compound trifluoromethanesulfonic acid or an equivalent compound resulting in most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine) cleavage while maintaining the integrity of the antibody. Chemical deglycosylation is described, for example, by Hakimuddin et al., Arch Biochem Biophys 259:52 (1987) and Edge et al., Anal Biochem 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on antibodies can be achieved by any of a number of endoglycosidases and exoglycosidases, eg, as described by Thotakura et al., Meth Enzymol 138:350 (1987).
另一类型的抗体共价修饰包括依据US专利号4,640,835、4,496,689、4,301,144、4,670,417、4,791,192或4,179,337所述方式将该抗体连接到许多非蛋白质聚合物例如聚乙二醇、聚丙二醇或聚氧化烯类(polyoxylalkylene)的一种。Another type of covalent modification of antibodies involves linking the antibody to a number of nonproteinaceous polymers such as polyethylene glycol, polypropylene glycol, or polyoxyalkylenes in the manner described in US Pat. (polyoxylalkylene) a kind of.
获得突变体或突变蛋白质的另一种优选技术是籍噬菌体展示进行亲和力成熟(Hawkins等人,J Mol Biol 254:889-896(1992)和Lowman等人,Biochemistry 30(45):10832-10838(1991))。简而言之，几个高变区位点(例如6-7个位点)被突变以产生每个位点所有可能的氨基酸取代。依此得到的抗体突变体作为与噬菌体颗粒上的蛋白的融合以单价形式被展示于噬菌体颗粒上。表现多种突变体的噬菌体可通过以下循环：多轮结合选择，随后对展示出高亲和力的突变体而进行分离和测序。Another preferred technique for obtaining mutant or mutated proteins is affinity maturation by phage display (Hawkins et al., J Mol Biol 254:889-896 (1992) and Lowman et al., Biochemistry 30(45):10832-10838( 1991)). Briefly, several hypervariable region positions (eg, 6-7 positions) are mutated to generate all possible amino acid substitutions at each position. The antibody mutants thus obtained are displayed in monovalent form on phage particles as fusions to proteins on the phage particles. Phage expressing multiple mutants can be passed through cycles of multiple rounds of selection for binding, followed by isolation and sequencing of mutants displaying high affinity.
选择新结合多肽的方法可使用结构相关多肽的库。通过诱变产生与例如噬菌体外壳蛋白融合的结构相关多肽的库，并将其展示在颗粒表面。随后这些颗粒与靶分子接触，将对靶具有最高亲和力的颗粒与具有低亲和力的颗粒分离出来。随后通过感染适宜的细菌宿主扩增高亲和力的结合物，并重复竞争性结合步骤。重复该过程，直至得到所需亲和力的多肽。Methods for selecting novel binding polypeptides may use libraries of structurally related polypeptides. Libraries of structurally related polypeptides fused to eg phage coat proteins are generated by mutagenesis and displayed on particle surfaces. These particles are then contacted with target molecules, separating those with the highest affinity for the target from those with lower affinity. High affinity binders are then amplified by infection of a suitable bacterial host and the competitive binding step repeated. This process is repeated until a polypeptide of the desired affinity is obtained.
或者，还可使用多价噬菌体(McCafferty等人(1990)Nature 348:552-554和Clackson等人(1991)Nature 352:624-628)来表达随机点突变(例如籍使用易错DNA聚合酶生成)，以生成噬菌体抗体片段的库，并随后筛选该库对IL-4和/或IL-13的亲和力(Hawkins等人(1992)J Mol Biol 254:889-896)。Alternatively, multivalent phages (McCafferty et al. (1990) Nature 348:552-554 and Clackson et al. (1991) Nature 352:624-628) can also be used to express random point mutations (e.g., by using error-prone DNA polymerases to generate ) to generate a library of phage antibody fragments and subsequently screen the library for affinity to IL-4 and/or IL-13 (Hawkins et al. (1992) J Mol Biol 254:889-896).
优选情况下，在亲和力成熟过程中，可复制的表达载体处于转录调节元件的严密调控下，调节培养条件使得展示一个以上融合蛋白拷贝的颗粒的量或数目小于约1％。同样在优选情况下，展示一个以上融合蛋白拷贝的颗粒的量小于展示单个融合蛋白拷贝的颗粒量的10％。优选情况下该量小于20％。Preferably, during affinity maturation, replicable expression vectors are under the tight control of transcriptional regulatory elements and culture conditions are adjusted such that the amount or number of particles displaying more than one copy of the fusion protein is less than about 1%. Also preferably, the amount of particles displaying more than one copy of the fusion protein is less than 10% of the amount of particles displaying a single copy of the fusion protein. Preferably the amount is less than 20%.
通过互换构架区或源自多个抗体的复合FR内不同抗体链的不同CDR，可产生功能等效物。因此，例如，对于一组给定的CDRs而言，通过取代不同的重链例如IgG1-4,、IgM、IgA1-2或IgD，不同种类的抗体有可能生成不同的IL-4和/或IL-13抗体类型和同种型。与之类似，通过将一组给定CDRs包埋入整个合成的构架区内，可产生位于本发明范围内的人工抗体。Functional equivalents can be generated by interchanging the framework regions or different CDRs of different antibody chains within a composite FR derived from multiple antibodies. Thus, for example, for a given set ofCDRs , it is possible for different classes of antibodies to generate different IL-4 and/or or IL-13 antibody type and isotype. Similarly, artificial antibodies within the scope of the invention can be produced by embedding a given set of CDRs throughout a synthetic framework region.
本发明的抗体片段和功能等效物包括其特异结合IL-4和/或IL-13的程度可被检测的分子。结合的可检测程度包括目的抗体结合能力的至少10-100％范围内的所有值，优选为至少50％、60％或70％，更优选为至少75％、80％、85％、90％、95％或99％。具有大于目的抗体100％亲和力的等效物也包括在内。Antibody fragments and functional equivalents of the invention include molecules for which the extent to which specific binding to IL-4 and/or IL-13 can be measured. The detectable degree of binding includes all values in the range of at least 10-100% of the binding capacity of the antibody of interest, preferably at least 50%, 60% or 70%, more preferably at least 75%, 80%, 85%, 90%, 95% or 99%. Equivalents with greater than 100% affinity for the antibody of interest are also included.
一般CDRs对于表位识别和抗体结合是重要的。但是，可对包含CDRs的残基进行变动，而不干扰抗体识别和结合关联表位的能力。例如，可进行不影响表位识别但却增加抗体对表位的结合亲和力的变动。基于对主要抗体序列的了解，几项研究调查了向抗体序列多个位点引入一个或多个氨基酸变动后对抗体性质的效应，例如结合和表达水平(Yang等人,1995,J Mol Biol 254:392-403；Rader等人,1998,Proc Natl Acad Sci USA 95:8910-8915；和Vaughan等人,1998,Nature Biotechnology 16,535-539)。Generally CDRs are important for epitope recognition and antibody binding. However, changes in the residues comprising the CDRs can be made without interfering with the ability of the antibody to recognize and bind the cognate epitope. For example, changes that do not affect epitope recognition but increase the binding affinity of the antibody for the epitope can be made. Based on what is known about the primary antibody sequence, several studies have investigated the effect on antibody properties, such as binding and expression levels, of introducing one or more amino acid changes at various positions in the antibody sequence (Yang et al., 1995, J Mol Biol 254 :392-403; Rader et al., 1998, Proc Natl Acad Sci USA 95:8910-8915; and Vaughan et al., 1998, Nature Biotechnology 16, 535-539).
因此，使用诸如寡核苷酸介导的定点诱变、盒诱变、易错PCR、DNA改组或大肠杆菌的突变株等方法改变CDR1、CDR2或CDR3或构架区中的重链和轻链基因的序列，可生成目的抗体的等效物(Vaughan等人,1998,Nat Biotech 16:535-539；和Adey等人,1996,Chap.16,pp.277-291,in Phage Display of Peptides and Proteins,eds.Kay等人,AcademicPress)。改变初级抗体核酸序列的方法可导致抗体的亲和力提高(Gram等人,1992,ProcNatl Acad Sci USA 89:3576-3580；Boder等人,2000,Proc Natl Acad Sci USA 97:10701-10705；Davies&Riechmann,1996,Immunotech 2:169-179；Thompson等人,1996,JMol Biol 256:77-88；Short等人,2002,J Biol Chem 277:16365-16370；和Furukawa等人,2001,J Biol Chem 276:27622-27628)。Therefore, alter the heavy and light chain genes in CDR1, CDR2, or CDR3 or framework regions using methods such as oligonucleotide-mediated site-directed mutagenesis, cassette mutagenesis, error-prone PCR, DNA shuffling, or mutant strains of E. coli Sequences that can generate equivalents of the antibody of interest (Vaughan et al., 1998, Nat Biotech 16:535-539; and Adey et al., 1996, Chap.16, pp.277-291, in Phage Display of Peptides and Proteins , eds. Kay et al., Academic Press). The method of changing the nucleic acid sequence of the primary antibody can lead to an increase in the affinity of the antibody (Gram et al., 1992, Proc Natl Acad Sci USA 89:3576-3580; Boder et al., 2000, Proc Natl Acad Sci USA 97:10701-10705; Davies & Riechmann, 1996 , Immunotech 2:169-179; Thompson et al., 1996, J Mol Biol 256:77-88; Short et al., 2002, J Biol Chem 277:16365-16370; and Furukawa et al., 2001, J Biol Chem 276:27622 -27628).
通过例如选择历经多轮选择而选择的多个氨基酸变化，可使用重复数轮的"多肽选择"以选择越来越高的亲和力结合。第一轮选择后可对配体的其它区域或氨基酸进行其它轮的选择，其中第一轮选择涉及配体或抗体多肽的氨基酸选择的首个区域。重复多轮选择，直到实现所需的亲和力性质。Repeated rounds of "polypeptide selection" can be used to select for increasingly higher affinity binding by, for example, selecting for multiple amino acid changes over multiple rounds of selection. Additional rounds of selection may be performed on other regions or amino acids of the ligand following the first round of selection involving the ligand or the first region of amino acid selection of the antibody polypeptide. Multiple rounds of selection are repeated until the desired affinity properties are achieved.
改良抗体还包括具有改良特征的抗体，它们籍动物免疫、杂交瘤形成和选择具有特异特征的抗体的标准技术而制备。Improved antibodies also include antibodies with improved characteristics prepared by standard techniques of animal immunization, hybridoma formation and selection of antibodies with specific characteristics.
"拮抗剂"系指能够抑制靶分子一种或多种生物活性(例如籍IL-4和/或IL-13的信号转导)的分子。拮抗剂通过灭活或杀灭被配体活化的细胞和/或干扰受体或配体活化(例如酪氨酸激酶活化)或配体结合到受体后的信号转导，可干扰受体结合到配体且反之亦然。所述拮抗剂可完全阻断受体-配体的相互作用，或实质降低这种相互作用。"Antagonist" refers to a molecule capable of inhibiting one or more biological activities of a target molecule (eg, signaling through IL-4 and/or IL-13). Antagonists interfere with receptor binding by inactivating or killing cells activated by the ligand and/or interfering with receptor or ligand activation (eg, tyrosine kinase activation) or signaling following ligand binding to the receptor to the ligand and vice versa. Such antagonists can completely block receptor-ligand interactions, or substantially reduce such interactions.
"激动剂"系指包括活化IL-4和/或IL-13一种或多种生物活性的蛋白质、多肽、肽、抗体、抗体片段、缀合物、大分子、小分子的化合物。激动剂可通过作为被配体活化的细胞的促分裂原作用和/或通过在配体结合到受体后干扰细胞失活或信号转导抑制，从而与受体对配体的结合相互作用，反之亦然。出于本发明的目的，激动剂的所有此类干涉位置均被视为相当的。"Agonist" refers to compounds including proteins, polypeptides, peptides, antibodies, antibody fragments, conjugates, macromolecules, and small molecules that activate one or more biological activities of IL-4 and/or IL-13. Agonists may interact with receptor-to-ligand binding by acting as mitogens for cells activated by the ligand and/or by interfering with cell inactivation or signal transduction inhibition following binding of the ligand to the receptor, vice versa. All such interfering positions of agonists are considered equivalent for the purposes of the present invention.
术语"细胞"、"细胞系"和"细胞培养物"包括它们的后代。还应理解，由于有意或无意的突变，所有的后代不一定精确相同，例如在DNA含量上。包括具有相同目的功能或生物性质的变体后代，其中目的功能或生物性质为针对原始细胞进行筛选。The terms "cell", "cell line" and "cell culture" include their progeny. It should also be understood that all progeny may not necessarily be exactly the same, eg, in DNA content, due to deliberate or inadvertent mutations. Included are variant progeny having the same desired function or biological property that was screened against the original cell.
术语"载体"意为核酸建构体、载体，其含有核酸、转基因、外源基因或目的基因，它们被有效地连接到适宜的调控序列以在适宜的宿主内进行转基因的表达。这类调控序列包括例如影响转录的启动子、控制转录的任选操纵子序列、编码适宜mRNA核糖体结合位点的序列以及调控转录和翻译终止的序列。载体可为质粒、噬菌体颗粒或仅仅是潜在的基因组插入物。一旦被转化到适宜的宿主体内，载体可独立于宿主的基因组进行复制和发挥功能，或在某些情形下可整合到宿主细胞的基因组上。在本说明书中，"质粒"和"载体"可互换使用，因为质粒是载体常见的使用形式。但是，本发明旨在包括发挥等效载体功能的此类其它形式的载体，它们为或正为本领域所知晓，例如病毒、携带核酸的合成分子、脂质体等。The term "vector" means a nucleic acid construct, a vector, which contains a nucleic acid, transgene, foreign gene or gene of interest, which are operably linked to appropriate regulatory sequences for expression of the transgene in a suitable host. Such regulatory sequences include, for example, a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding a suitable mRNA ribosomal binding site, and sequences that regulate termination of transcription and translation. Vectors can be plasmids, phage particles or simply potential genomic inserts. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases can integrate into the genome of the host cell. In this specification, "plasmid" and "vector" are used interchangeably, since plasmids are the commonly used form of vectors. However, the invention is intended to include such other forms of vectors, which are or are known in the art, such as viruses, synthetic nucleic acid-carrying molecules, liposomes, etc., which function as equivalent vectors.
就治疗目的而言"哺乳动物"系指可被归类为哺乳动物的任何动物，其包括人、家畜和农场动物、非人灵长类动物和动物园动物、竞技动物或宠物，例如狗、马、猫、牛等。"Mammal" for therapeutic purposes means any animal that can be classified as a mammal, including humans, domestic and farm animals, non-human primates and zoo animals, sport animals or pets such as dogs, horses , cats, cows, etc.
可在本文所述或本领域知晓的测定中筛选或使用目的抗体。这类测定通常需要可检测如被标记的试剂。本文所使用的词语"标记"系指可直接或间接缀合到分子或蛋白例如抗体上的可检测化合物或组合物。标记可本身为可检测的(例如放射性同位素标记、颗粒或荧光标记)，或就酶标记而言可催化能被检测的底物化合物或组合物的化学改变。Antibodies of interest can be screened for or used in assays described herein or known in the art. Such assays generally require detectable, eg labeled, reagents. The word "label" as used herein refers to a detectable compound or composition that can be directly or indirectly conjugated to a molecule or protein, such as an antibody. Labels can be detectable per se (eg, radioisotopic labels, particles, or fluorescent labels), or in the case of enzymatic labels can catalyze a chemical alteration of a substrate compound or composition that can be detected.
正如本文所使用，"固相"意为实体或分子例如本发明所述抗体可粘附其上的非水基质。本文所包括的固相实例包括部分或全部由玻璃(例如可控孔径玻璃)、多糖(例如琼脂糖)、聚丙烯酰胺、聚苯乙烯、聚乙烯醇和聚硅氧烷形成的固相。在一些实施方案中，根据上下文，所述固相可包括测定板的孔；在其它情况下可用于纯化柱(例如亲和力层析柱)。因此，所述固相可以是纸、珠、塑料、芯片等，可从许多材料例如硝化纤维素、琼脂糖、聚苯乙烯、聚丙烯、硅等制备，还可采取许多构象。As used herein, "solid phase" means a non-aqueous matrix to which entities or molecules, such as antibodies of the invention, may adhere. Examples of solid phases contemplated herein include solid phases formed in part or in whole of glass (eg, controlled pore glass), polysaccharides (eg, agarose), polyacrylamide, polystyrene, polyvinyl alcohol, and polysiloxane. In some embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other cases it may be used in a purification column (eg, an affinity chromatography column). Thus, the solid phase can be paper, beads, plastic, chips, etc., can be prepared from many materials such as nitrocellulose, agarose, polystyrene, polypropylene, silicon, etc., and can adopt many conformations.
依据为本领域技术人员熟知的方法且例如参见下文，本领域业已知晓编码IL-4和IL-13的基因或cDNA，它们可被克隆到质粒或其它表达载体，并在众多表达体系的任意一种中进行表达。Genes or cDNAs encoding IL-4 and IL-13 are known in the art and can be cloned into plasmids or other expression vectors and expressed in any of a number of expression systems according to methods well known to those skilled in the art and see, for example, below. expressed in the species.
编码氨基酸序列突变体的核酸序列可通过许多本领域知晓的方法进行制备。这些方法包括但不限于先前制备的目的分子的突变体或非突变体形式的寡核苷酸-介导(或定点)诱变、PCR诱变和盒诱变(参见例如Kunkel,Proc Natl Acad Sci USA 82:488(1985))。Nucleic acid sequences encoding amino acid sequence mutants can be prepared by many methods known in the art. These methods include, but are not limited to, oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared mutant or non-mutant forms of the molecule of interest (see, e.g., Kunkel, Proc Natl Acad Sci. USA 82:488 (1985)).
本发明抗体或其片段、衍生物或类似物(例如本发明抗体的重链或轻链、本发明的单链抗体或本发明的抗体突变蛋白质)的重组表达包括构建含有编码本文所述的抗体或抗体片段的多核苷酸的表达载体。一旦获得编码抗体分子的多核苷酸，可通过本领域知晓的重组DNA技术产生用于生产所述抗体的载体。构建含有抗体编码序列和适宜转录和翻译调控信号的表达载体。这些方法包括例如体外重组DNA技术、合成技术和体内基因重组。Recombinant expression of an antibody of the invention or a fragment, derivative or analog thereof (e.g., a heavy or light chain of an antibody of the invention, a single chain antibody of the invention, or an antibody mutein of the invention) involves constructing or antibody fragment polynucleotide expression vector. Once a polynucleotide encoding an antibody molecule is obtained, vectors for production of the antibody can be generated by recombinant DNA techniques known in the art. An expression vector is constructed containing the antibody coding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo genetic recombination.
通过常规技术将表达载体转移到宿主细胞体内，随后通过常规技术培养被转染的细胞，以产生本发明的抗体或片段。在本发明的一个方面，编码重链和轻链的载体可在宿主细胞内共表达，以表达本文详述的整个免疫球蛋白分子。The expression vector is transferred into host cells by conventional techniques, and the transfected cells are then cultured by conventional techniques to produce the antibodies or fragments of the present invention. In one aspect of the invention, vectors encoding the heavy and light chains can be co-expressed in a host cell to express the entire immunoglobulin molecule detailed herein.
可使用许多宿主/表达载体体系来表达本发明的抗体分子。这些表达体系不仅代表可产生和随后纯化目的编码序列的运载体(vehicle)，还代表了以适宜核苷酸编码序列转化或转染时可原位表达本发明抗体分子的细胞。细菌细胞例如大肠杆菌和真核细胞常用于重组抗体分子的表达，尤其是用于全长重组抗体分子的表达。例如，哺乳动物细胞例如CHO细胞与例如携带来自人巨细胞病毒的主要中间体即时早期基因启动子元件的载体的载体联合是抗体的有效表达体系(Foecking等人,Gene 45:101(1986)；和Cockett等人,Bio/Technology 8:2(1990))。正如本领域所知晓，还可使用植物和植物细胞培养物、昆虫细胞等来生产目的蛋白。A number of host/expression vector systems can be used to express the antibody molecules of the invention. These expression systems represent not only vehicles that can produce and subsequently purify the coding sequence of interest, but also cells that can express the antibody molecule of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequence. Bacterial cells such as E. coli and eukaryotic cells are commonly used for the expression of recombinant antibody molecules, especially for the expression of full-length recombinant antibody molecules. For example, mammalian cells such as CHO cells in combination with a vector such as a vector carrying a major intermediate immediate early gene promoter element from human cytomegalovirus is an efficient expression system for antibodies (Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio/Technology 8:2 (1990)). Plants and plant cell cultures, insect cells, etc. can also be used to produce the protein of interest, as is known in the art.
此外，选择可调节插入序列表达或以所需的特异方式修饰和加工基因产物的宿主细胞。蛋白产物的这些修饰(例如糖基化)和加工(例如切割)对于蛋白功能可能是重要的。不同的宿主细胞具有蛋白质及基因产物翻译后加工和修饰的性质和特异机制。可选择适宜的细胞系或宿主体系，以确保所表达的目的抗体的正确修饰和加工。因此，可使用拥有用于主要转录物适宜加工、基因产物糖基化和磷酸化的细胞机制的真核宿主细胞。这些哺乳动物宿主细胞包括但不限于CHO、COS、293、3T3或骨髓瘤细胞。In addition, host cells are selected that regulate the expression of the inserted sequence or that modify and process the gene product in the specific manner desired. These modifications (eg, glycosylation) and processing (eg, cleavage) of protein products may be important for protein function. Different host cells have properties and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be selected to ensure correct modification and processing of the expressed antibody of interest. Thus, eukaryotic host cells possessing the cellular machinery for appropriate processing of primary transcripts, glycosylation and phosphorylation of gene products can be used. These mammalian host cells include, but are not limited to, CHO, COS, 293, 3T3 or myeloma cells.
稳定表达对于长期高产率生产重组蛋白而言是优选的。例如可改造稳定表达抗体分子的细胞系。与其使用含有病毒复制起点的表达载体，不如使用受适宜表达调控元件(例如启动子、增强子、序列、转录终止子、多聚腺苷酸位点等)调控的DNA和选择标记转化宿主细胞。引入外来DNA后，可让改造细胞在富集培养基上生长1至2天，随后将其转移至选择性培养基上。重组质粒的选择性标记赋予对选择的抗性，使得细胞将质粒稳定整合到染色体上，并扩展成细胞系。这些改造的细胞系不仅可用于抗体制备，还可用于筛选和评估直接或间接与抗体分子相互作用的化合物。Stable expression is preferred for long-term high-yield production of recombinant proteins. For example, cell lines can be engineered to stably express antibody molecules. Rather than using an expression vector containing a viral origin of replication, host cells are transformed with DNA and a selectable marker regulated by appropriate expression control elements (eg, promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After the introduction of foreign DNA, the engineered cells can be grown on enriched media for 1 to 2 days before being transferred to selective media. The selectable marker of the recombinant plasmid confers resistance to selection, allowing the cells to stably integrate the plasmid into the chromosome and expand into cell lines. These engineered cell lines can be used not only for antibody production, but also for screening and evaluating compounds that directly or indirectly interact with antibody molecules.
可使用许多选择体系，其分别包括但不限于单纯疱疹病毒胸苷激酶(Wigler等人,Cell 11:223(1977))、次黄嘌呤-鸟嘌呤磷酸核糖转移酶(Szybalska等人,Proc Natl AcadSci USA 48:202(1992))、谷氨酸合成酶在甲硫氨酸砜亚胺的存在下选择(Adv Drug DelRev 58,671,2006并参看Lonza Group Ltd.的网站或文献)以及tk、hgprt或aprt细胞中的腺嘌呤磷酸核糖转移酶基因(Lowy等人,Cell 22:817(1980))。同样，可使用抗代谢物抗性作为选择下列基因的基础：dhfr，其赋予甲胺蝶呤抗性(Wigler等人,Proc Natl Acad SciUSA 77:357(1980)；O'Hare等人,Proc Natl Acad Sci USA 78:1527(1981))；gpt，其赋予霉酚酸抗性(Mulligan等人,Proc Natl Acad Sci USA 78:2072(1981))；neo，其赋予氨基糖苷G-418抗性(Wu等人,Biotherapy 3:87(1991))；以及hygro，其赋予潮霉素抗性(Santerre等人,Gene 30:147(1984))。可常规应用重组DNA技术领域知晓的方法来选择所需的重组克隆，这些方法的描述见于例如Ausubel等人,eds.,Current Protocols inMolecular Biology,John Wiley&Sons(1993)；Kriegler,Gene Transfer andExpression,A Laboratory Manual,Stockton Press(1990)；Dracopoli等人,eds.,Current Protocols in Human Genetics,John Wiley&Sons(1994)；和Colberre-Garapin等人,J Mol Biol 150:1(1981)。A number of selection systems can be used including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl AcadSci. USA 48:202 (1992)), glutamate synthase selection in the presence of methionine sulfoximine (Adv Drug DelRev 58,671, 2006 and see Lonza Group Ltd. website or literature) and tk, hgprt or aprt Adenine phosphoribosyltransferase gene in cells (Lowy et al., Cell 22:817 (1980)). Likewise, antimetabolite resistance can be used as the basis for selection of the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77:357 (1980); O'Hare et al., Proc Natl Acad Sci USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 ( Wu et al., Biotherapy 3:87 (1991)); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)). Desired recombinant clones can be selected routinely using methods known in the art of recombinant DNA technology and described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press (1990); Dracopoli et al., eds., Current Protocols in Human Genetics, John Wiley & Sons (1994); and Colberre-Garapin et al., J Mol Biol 150:1 (1981).
通过载体扩增可增加抗体分子的表达水平(例如参见Bebbington等人,DNACloning,Vol.3.Academic Press(1987))。当表达抗体的载体体系中的标记物可扩增时，培养物中抑制剂水平的升高会增加标记物基因的拷贝数目。由于被扩增区域与抗体基因有关联，抗体的产生也会上升(Crouse等人,Mol Cell Biol 3:257(1983))。Expression levels of antibody molecules can be increased by vector amplification (see, eg, Bebbington et al., DNA Cloning, Vol. 3. Academic Press (1987)). When the marker is amplifiable in the vector system expressing the antibody, increased levels of the inhibitor in the culture will increase the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production is also increased (Crouse et al., Mol Cell Biol 3:257 (1983)).
可使用本发明的两种或更多表达载体对宿主细胞进行共转染，例如第一载体编码源自重链的多肽，而第二载体编码源自轻链的多肽。这两个载体可含有相同的选择标记物，这些标记物可使重链和轻链多肽的表达相同。或者，可使用编码并可表达重链和轻链多肽的单个载体。在这种情况下，轻链应置于重链之前，以避免产生过多不含毒性的重链(Proudfoot,Nature 322:52(1986)；和Kohler,Proc Natl Acad Sci USA 77:2197(1980))。重链和轻链的编码序列可包括cDNA或基因组DNA。Host cells can be co-transfected with two or more expression vectors of the invention, eg, a first vector encoding a heavy chain-derived polypeptide and a second vector encoding a light chain-derived polypeptide. The two vectors may contain the same selectable markers which allow for the same expression of the heavy and light chain polypeptides. Alternatively, a single vector that encodes and expresses both heavy and light chain polypeptides can be used. In this case, the light chain should be placed ahead of the heavy chain to avoid excess non-toxic heavy chain (Proudfoot, Nature 322:52 (1986); and Kohler, Proc Natl Acad Sci USA 77:2197 (1980 )). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
一旦通过动物、化学合成或重组表达产生本发明的抗体分子，可通过本领域知晓的任何纯化免疫球蛋白分子的方法例如层析(例如离子交换、亲和层析等，尤其是经蛋白质A和大小排阻层析后对IL-4和/或IL-13的亲和层析)、离心、差异溶解度或通过蛋白纯化的任何其它标准技术纯化抗体分子。此外，本发明所述抗体或抗体片段可被融合到本文所述的异源多肽序列或本领域知晓的其它多肽序列，以便于纯化。Once the antibody molecules of the invention have been produced by animal, chemical synthesis or recombinant expression, immunoglobulin molecules can be purified by any method known in the art such as chromatography (e.g. ion exchange, affinity chromatography, etc., especially by protein A and Antibody molecules are purified by size exclusion chromatography followed by affinity chromatography for IL-4 and/or IL-13), centrifugation, differential solubility, or by any other standard technique for protein purification. In addition, antibodies or antibody fragments of the invention may be fused to heterologous polypeptide sequences described herein or other polypeptide sequences known in the art to facilitate purification.
可通过本领域知晓的任何适宜方法生成本发明所述抗体。本发明所述抗体可包括多克隆抗体，尽管由于抗体修饰以优化其在人体内的使用以及优化抗体本身的使用，但单克隆抗体仍是优选的，这是因其生产和操纵特定蛋白方便。制备多克隆抗体的方法为本领域技术人员所知晓(Harlow等人,Antibodies:a Laboratory Manual,Cold Spring HarborLaboratory Press,2nd ed.(1988))。Antibodies of the invention can be produced by any suitable method known in the art. The antibodies of the invention may include polyclonal antibodies, although monoclonal antibodies are preferred due to the ease of production and manipulation of specific proteins, although the antibodies are modified to optimize their use in the human body as well as to optimize the use of the antibodies themselves. Methods for preparing polyclonal antibodies are known to those skilled in the art (Harlow et al., Antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988)).
本发明所述抗体优选包括单克隆抗体。可使用杂交瘤技术例如由Kohler等人,Nature 256:495(1975)；US专利号4,376,110；Harlow等人,Antibodies:A LaboratoryManual,Cold Spring Harbor Laboratory Press,2nd ed.(1988)和Hammerling等人,Monoclonal Antibodies and T-Cell Hybridomas,Elsevier(1981)描述的、重组DNA方法例如制备和使用转染瘤的方法或为技术人员知晓的其它方法制备单克隆抗体。可用于产生单克隆抗体的方法的其它实例包括但不限于人B细胞杂交瘤技术(Kosbor等人,ImmunologyToday 4:72(1983)；和Cole等人,Proc Natl Acad Sci USA 80:2026(1983))和EBV-杂交瘤技术(Cole等人，Monoclonal Antibodies and Cancer Therapy,pp.77-96,Alan R.Liss(1985))。这些抗体可为包括IgG、IgM、IgE、IgA和IgD的任何免疫球蛋白类别和其任何亚类的抗体。可在体外或体内培养产生本发明mAb的杂交瘤。The antibodies of the present invention preferably comprise monoclonal antibodies. Hybridoma technology can be used, for example, by Kohler et al., Nature 256:495 (1975); US Pat. No. 4,376,110; Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2nd ed. (1988) and Hammerling et al., Monoclonal Antibodies and T-Cell Hybridomas, described by Elsevier (1981), recombinant DNA methods such as methods of making and using transfectomas, or other methods known to the skilled person to prepare monoclonal antibodies. Other examples of methods that can be used to generate monoclonal antibodies include, but are not limited to, human B-cell hybridoma technology (Kosbor et al., Immunology Today 4:72 (1983); and Cole et al., Proc Natl Acad Sci USA 80:2026 (1983) ) and EBV-hybridoma technology (Cole et al., Monoclonal Antibodies and Cancer Therapy, pp.77-96, Alan R. Liss (1985)). These antibodies may be antibodies of any immunoglobulin class, including IgG, IgM, IgE, IgA and IgD, and any subclass thereof. Hybridomas producing mAbs of the invention can be cultured in vitro or in vivo.
在杂交瘤模型中，对宿主例如小鼠、人源化小鼠、携带人免疫系统基因的转基因小鼠、仓鼠、兔、大鼠、骆驼或其它任何适宜的宿主动物进行免疫，以引发产生或能够产生特异结合IL-4或IL-13的抗体的淋巴细胞。可选地，在体外免疫篱笆细胞。使用适宜的融合剂例如聚乙二醇使淋巴细胞随后与骨髓瘤细胞融合，形成杂交瘤细胞(Goding,MonoclonalAntibodies:Principles and Practice,Academic Press,pp.59-103(1986))。In the hybridoma model, hosts such as mice, humanized mice, transgenic mice carrying human immune system genes, hamsters, rabbits, rats, camels, or any other suitable host animal are immunized to elicit production or Lymphocytes capable of producing antibodies that specifically bind IL-4 or IL-13. Alternatively, fence cells are immunized in vitro. Lymphocytes are then fused with myeloma cells using a suitable fusing agent such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59-103 (1986)).
一般而言，制备产生抗体的杂交瘤时，如需要人来源细胞时可使用外周血淋巴细胞("PBL")，或如需要非人哺乳动物来源时则可使用脾细胞或淋巴结细胞。永生细胞系通常是转化的哺乳动物细胞，尤其是啮齿类、牛或人来源的骨髓瘤细胞。一般使用大鼠或小鼠的骨髓瘤细胞系。可在适宜的培养基上培养杂交瘤细胞，该培养基优选含有可抑制未融合的永生细胞的生长或生存的一种或多种物质。例如，如果亲代细胞缺乏次黄嘌呤鸟嘌呤磷酸核糖转移酶(HGPRT或HPRT)，用于杂交瘤的培养基一般会含有次黄嘌呤、氨蝶呤和胸苷("HAT培养基")，它们是防止缺乏HGPRT的细胞生长的物质。In general, antibody-producing hybridomas are prepared using peripheral blood lymphocytes ("PBL") if cells of human origin are desired, or spleen cells or lymph node cells if non-human mammalian sources are desired. Immortal cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine or human origin. Typically rat or mouse myeloma cell lines are used. Hybridoma cells may be cultured in a suitable medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortal cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), culture medium for hybridomas will generally contain hypoxanthine, aminopterin, and thymidine ("HAT medium"), which Is a substance that prevents the growth of cells lacking HGPRT.
优选的永生细胞系是有效融合并支持所选择的产生抗体的细胞稳定高水平产生抗体的细胞系，它们对培养基例如HAT培养基敏感。这些骨髓瘤细胞系中有鼠骨髓瘤系，例如源自可从Salk Institute Cell Distribution Center,San Diego,Calif获得的MOPC-21和MPC-11小鼠肿瘤和可从美国典型培养物保藏中心(American Type CultureCollection),Manassas,VA获得的SP2/0、FO或X63-Ag8-653细胞的细胞系。Preferred immortal cell lines are those that fuse efficiently and support stable high-level production of antibody by selected antibody-producing cells, which are sensitive to a medium such as HAT medium. Among these myeloma cell lines are murine myeloma lines derived, for example, from the MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif and from the American Type Culture Collection (American Type Culture Collection). Type Culture Collection), Manassas, VA obtained cell lines of SP2/0, FO or X63-Ag8-653 cells.
还描述了人骨髓瘤和小鼠-人异骨髓瘤(heteromyeloma)细胞系用于产生人单克隆抗体(Kozbor,J Immunol 133:3001(1984)；和Brodeur等人,Monoclonal AntibodyProduction Techniques and Applications,Marcel Dekker,Inc,pp.51-63(1987))。51-63(1987))。还可使用小鼠骨髓瘤细胞系NSO(European Collection of Cell Cultures,Salisbury,Wilshire,UK)。Human myeloma and mouse-human heteromyeloma (heteromyeloma) cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J Immunol 133:3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc, pp. 51-63 (1987)). 51-63 (1987)). The mouse myeloma cell line NSO (European Collection of Cell Cultures, Salisbury, Wilshire, UK) can also be used.
另外一种选择是使用电融合而非化学融合来形成杂交瘤。不使用融合，而是使用例如Epstein Barr病毒或其它转化基因来永生化B细胞，例如参见Zurawaki等人,Monoclonal Antibodies,编辑，Kennett等人,Plenum Press,pp.19-33.(1980)。19-33.(1980)。还可使用表达免疫球蛋白的转基因小鼠和移植有人B淋巴细胞的重症联合免疫缺陷(severe combined immunodeficient，SCID)小鼠。Another option is to use electrofusion rather than chemical fusion to form hybridomas. Instead of using fusion, B cells are immortalized using eg Epstein Barr virus or other transforming genes, see eg Zurawaki et al., Monoclonal Antibodies, ed., Kennett et al., Plenum Press, pp. 19-33. (1980). 19-33. (1980). Transgenic mice expressing immunoglobulins and severe combined immunodeficient (SCID) mice transplanted with human B lymphocytes can also be used.
测定生长有杂交瘤细胞的培养基中的针对IL-4和/或IL-13的单克隆抗体的产生。可通过免疫沉淀或通过体外结合测定例如放射免疫测定(RIA)、荧光细胞测量分析(flluorocytometric analysis,FACS)或酶连接免疫吸附测定(ELISA)测定杂交瘤细胞所产生的单克隆抗体的结合特异性。这些技术为本领域知晓，并在技术人员的技艺范围之内。可通过例如Scatchard分析(Munson等人,Anal Biochem 107:220(1980))测定单克隆抗体对IL-4和/或IL-13的结合亲和力。The production of monoclonal antibodies against IL-4 and/or IL-13 in media grown with hybridoma cells is assayed. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA), fluorescent cytometric analysis (FACS) or enzyme-linked immunosorbent assay (ELISA) . These techniques are known in the art and are within the skill of the artisan. The binding affinity of monoclonal antibodies to IL-4 and/or IL-13 can be determined by, eg, Scatchard analysis (Munson et al., Anal Biochem 107:220 (1980)).
鉴定产生所需特异性、亲和力和/或活性的抗体的杂交瘤细胞后，这些克隆可通过有限稀释方法进行亚克隆，并按标准方法(Goding,Monoclonal Antibodies:Principlesand Practice,Academic Press,pp.59-103(1986)进行培养。适宜的培养基包括例如Dulbecco改良的Eagle培养基(D-MEM)或RPMI-1640培养基。此外，杂交瘤细胞可作为腹水肿瘤在动物体内生长。After identification of hybridoma cells producing antibodies of desired specificity, affinity, and/or activity, these clones can be subcloned by limiting dilution methods and cloned according to standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, pp. 59 -103 (1986). Suitable media include, for example, Dulbecco's modified Eagle's medium (D-MEM) or RPMI-1640 medium. Alternatively, hybridoma cells can be grown in animals as ascites tumors.
通过常规免疫球蛋白纯化方法例如蛋白质A-Sepharose、蛋白质G-Sepharose、羟磷灰石层析、凝胶排阻层析、凝胶电泳、透析或亲和层析从培养基、腹水液体或血清中适宜分开或分离出经亚克隆分泌的单克隆抗体。From culture medium, ascitic fluid or serum by conventional immunoglobulin purification methods such as protein A-Sepharose, protein G-Sepharose, hydroxyapatite chromatography, gel exclusion chromatography, gel electrophoresis, dialysis or affinity chromatography suitable for isolating or isolating monoclonal antibodies secreted by subcloning.
本领域存在许多用于产生单克隆抗体的方法，因此本发明不局限于它们仅在杂交瘤中的制备。例如，可通过重组DNA方法例如US专利号4,816,567所述方法制备单克隆抗体。在这种情况下，术语"单克隆抗体"系指源自单个真核生物、噬菌体或原核生物克隆的抗体。Many methods exist in the art for the production of monoclonal antibodies and thus the invention is not limited to their production in hybridomas only. For example, monoclonal antibodies can be prepared by recombinant DNA methods such as those described in US Patent No. 4,816,567. In this context, the term "monoclonal antibody" refers to an antibody derived from a single eukaryotic, phage or prokaryotic clone.
使用常规方法(例如使用可特异结合编码鼠抗体重链和轻链或来自人、人源化或其它来源的此类链的基因的寡核苷酸探针)可轻易分离和测序编码本发明所述单克隆抗体的DNA(Innis等人,PCR Protocols.A Guide to Methods and Applications,Academic(1990)和Sanger等人,Proc Natl Acad Sci 74:5463(1977))。杂交瘤细胞作为这类DNA的来源。一旦分离，所述DNA可被置于表达载体中，随后该表达载体被转染到本来不产生免疫球蛋白的宿主细胞例如大肠杆菌细胞、NSO细胞、COS细胞、中国仓鼠卵巢(CHO)细胞或骨髓瘤细胞中，以在重组的宿主细胞体内获得单克隆抗体的合成。还可通过例如用人重链和轻链恒定结构域的编码序列取代同源鼠序列(US专利号4,816,567；和Morrison等人,ProcNatl Acad Sci USA 81:6851(1984))或通过将非免疫球蛋白多肽的全部或部分编码序列共价结合到免疫球蛋白编码序列，从而对所述DNA进行修饰。这些非免疫球蛋白多肽可被替换为本发明抗体的恒定结构域，或可被替换为本发明抗体的一个IL-4或IL-13结合位点的可变结构域，以形成嵌合的二价抗体。Genes encoding the compounds of the invention can be readily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that bind specifically to genes encoding the heavy and light chains of murine antibodies, or such chains from human, humanized, or other sources). DNA of monoclonal antibodies described above (Innis et al., PCR Protocols. A Guide to Methods and Applications, Academic (1990) and Sanger et al., Proc Natl Acad Sci 74:5463 (1977)). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulins such as E. coli cells, NSO cells, COS cells, Chinese Hamster Ovary (CHO) cells or In myeloma cells to obtain monoclonal antibody synthesis in recombinant host cells. Human heavy and light chain constant domain coding sequences may also be substituted for the homologous murine sequences, for example (US Pat. No. 4,816,567; and Morrison et al., ProcNatl Acad Sci USA 81:6851 (1984)) or by substituting the non-immunoglobulin protein All or part of the coding sequence for the polypeptide is covalently bound to the immunoglobulin coding sequence, thereby modifying the DNA. These non-immunoglobulin polypeptides can be replaced by the constant domains of the antibodies of the invention, or can be replaced by the variable domains of one of the IL-4 or IL-13 binding sites of the antibodies of the invention to form chimeric binary Antibodies.
这些抗体可为单价抗体。用于制备单价抗体的方法为本领域所熟知。例如，一种方法涉及免疫球蛋白轻链和修饰重链的重组表达。重链一般在Fc区的任意位置被截短，以防止重链交联。或者，相关半胱氨酸残基可被另一氨基酸残基取代或被缺失，以防止交联。These antibodies can be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated anywhere in theFc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residue can be substituted with another amino acid residue or deleted to prevent cross-linking.
可通过已知技术生成识别特异表位的抗体片段。传统上通过完整抗体的蛋白水解消化得到这些片段(例如参见Morimoto等人,J Biochem Biophys Methods 24:107(1992)；和Brennan等人,Science 229:81(1985))。例如使用酶如木瓜酶(产生Fab片段)或胃蛋白酶(产生F(ab')2片段)对免疫球蛋白分子进行蛋白水解切割，可产生本发明的Fab和F(ab')2片段。F(ab')2片段含有可变区、轻链恒定区和重链的CH1结构域。但是可直接通过重组的宿主细胞产生这些片段。例如，抗体片段可从抗体噬菌体库分离得到。或者，F(ab')2-SH片段可直接从大肠杆菌中回收并籍化学方式偶联形成F(ab’)2片段(Carter等人,Bio/Technology 10:163(1992)。根据另一方法，F(ab’)2片段可直接从重组的宿主细胞培养物中分离得到。产生抗体片段的其它技术对于本领域技术人员是显而易见的。在其它实施方案中，选择的抗体是单链Fv片段(Fv)(WO 93/16185)。Antibody fragments that recognize specific epitopes can be generated by known techniques. These fragments are traditionally obtained by proteolytic digestion of intact antibodies (see, eg, Morimoto et al., J Biochem Biophys Methods 24:107 (1992); and Brennan et al., Science 229:81 (1985)).Fab and F(ab')2 fragments of the invention can be produced, for example, by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (to produceFab fragments) or pepsin (to produce F(ab')2 fragments) . The F(ab')2 fragment contains the variable region, the constant region of the light chain, and theCH1 domain of the heavy chain. However, these fragments can be produced directly by recombinant host cells. For example, antibody fragments can be isolated from antibody phage libraries. Alternatively, F(ab')2 -SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163 (1992). According to another method, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain F Fragmentv (Fv ) (WO 93/16185).
对于某些用途包括人体内使用抗体和体外检测测定而言，可优选使用嵌合的、人源化或人抗体。产生嵌合抗体的方法为本领域所知晓，例如参见Morrison,Science 229:1202(1985)；Oi等人,BioTechniques 4:214(1986)；Gillies等人,J Immunol Methods125:191(1989)；和US专利号5,807,715；4,816,567以及4,816,397。For certain uses, including use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized or human antibodies. Methods for generating chimeric antibodies are known in the art, see, e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J Immunol Methods 125:191 (1989); and US Patent Nos. 5,807,715; 4,816,567 and 4,816,397.
人源化抗体源自在非人物种体内生成的可结合IL-4和/或IL-13的抗体分子，其中来自其的一个或多个CDRs被插入到来自人免疫球蛋白分子的FR区。使用许多本领域知晓的多种技术例如CDR移植(EPO 239,400；WO 91/09967；和US专利号5,225,539；5,530,101；以及5,585,089),贴面或表面重塑(EPO 592,106；EPO 519,596；Padlan,MolecularImmunology 28:489(1991)；Studnicka等人,Protein Engineering 7:805(1994)；和Roguska等人,Proc Natl Acad Sci USA 91:969(1994)),以及链改组(US专利号5,565,332)可人源化抗体。Humanized antibodies are derived from antibody molecules that bind IL-4 and/or IL-13 produced in a non-human species from which one or more CDRs have been inserted into FR regions derived from human immunoglobulin molecules. Using a variety of techniques known in the art such as CDR grafting (EPO 239,400; WO 91/09967; and US Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EPO 592,106; EPO 519,596; Padlan, Molecular Immunology 28 :489 (1991); Studnicka et al., Protein Engineering 7:805 (1994); and Roguska et al., Proc Natl Acad Sci USA 91:969 (1994)), and chain shuffling (US Pat. No. 5,565,332) can be humanized Antibody.
人源化抗体具有一个或多个来自非人来源的氨基酸残基。这些非人的氨基酸残基通常被称作"导入"残基，它们一般取自"导入"的可变结构域。依据Winter及其同事等人的方法(Jones等人,Nature 321:522(1986)；Riechmann等人,Nature 332:323(1988)和Verhoeyen等人,Science 239:1534(1988))通过以非人的CDR或CDRs序列的部分替换人抗体的相应序列，可基本上进行人源化。因此，这些"人源化"抗体是嵌合抗体(US专利号4,816,567)，其中实质小于完整人可变结构域的序列已被来自非人物种的相应序列所取代。实践中，人源化抗体一般是人抗体，其中一些CDRs残基和可能的一些FR残基被啮齿类动物抗体的类似位点所取代。重链恒定区和铰接区可来自任何类别或亚类以获得需要的效果，如一个特定的效应物功能。Humanized antibodies have one or more amino acid residues from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are generally taken from an "import" variable domain. According to the methods of Winter and colleagues et al. (Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988) and Verhoeyen et al., Science 239:1534 (1988)) by using non-human Substituting the CDRs or parts of the CDRs sequences for the corresponding sequences of human antibodies can be substantially humanized. Thus, these "humanized" antibodies are chimeric antibodies (US Patent No. 4,816,567) in which substantially less than an intact human variable domain sequence has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDRs and possibly some FR residues are substituted by analogous sites from rodent antibodies. The heavy chain constant and hinge regions can be from any class or subclass to achieve the desired effect, such as a particular effector function.
通常，人构架区的构架残基可被来自CDR供体抗体的相应残基取代，从而改变并有可能提高抗原结合。构架取代可籍本领域知晓方法进行鉴定，例如经建立CDR与构架残基相互作用的模型来鉴定对于抗原结合的重要构架残基，以及经序列比较以鉴定特定位置处的异常构架残基，例如参阅US专利号5,585,089和Riechmann等人,Nature 332:323(1988)。Often, framework residues of the human framework regions can be substituted by corresponding residues from the CDR donor antibody, thereby altering and possibly improving antigen binding. Framework substitutions can be identified by methods known in the art, such as by modeling the interactions of CDRs with framework residues to identify framework residues important for antigen binding, and by sequence comparison to identify unusual framework residues at specific positions, e.g. See US Patent No. 5,585,089 and Riechmann et al., Nature 332:323 (1988).
更优选地，人源化抗体保留对IL-4和/或IL-13的高亲和力，并且保留或获得其它有利的生物性质。因此，利用亲代序列和人源化序列的三维模型，通过分析亲代序列和多种概念性人源化产物的方法，制备成人源化抗体。三维免疫球蛋白模型通常可为本领域技术人员利用和熟知。可获得说明和展示所选择候选免疫球蛋白序列的可能三维构象结构的计算机程序。检查此展示允许分析某些残基在候选免疫球蛋白序列发挥功能中的可能作用，即分析影响候选免疫球蛋白结合IL-4和/或IL-13能力的残基。籍此种方式，可从接收和导入序列选择和合并FR残基，使得所需抗体特性得到最大化，例如增加的对靶抗原的亲和力，尽管是CDR残基直接并最实质性地影响IL-4和/或IL-13结合。还可修饰CDR区域，使其含有一个或多个与得自亲代抗体(CDR得自该亲代抗体)氨基酸不同的氨基酸，以提供增强或不同的目的性质，例如更大的亲和力或更大的亲合力(avidity)的结合。More preferably, humanized antibodies retain high affinity for IL-4 and/or IL-13 and retain or acquire other favorable biological properties. Therefore, using three-dimensional models of the parental and humanized sequences, humanized antibodies were prepared by analyzing the parental sequences and various conceptual humanized products. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of this display allows the analysis of the possible role of certain residues in the functioning of the candidate immunoglobulin sequence, ie the analysis of residues that affect the ability of the candidate immunoglobulin to bind IL-4 and/or IL-13. In this way, FR residues can be selected and combined from the receiving and importing sequences such that desired antibody properties are maximized, such as increased affinity for the target antigen, although it is the CDR residues that directly and most substantially affect IL- 4 and/or IL-13 binding. CDR regions can also be modified to contain one or more amino acids that differ from the amino acids from the parent antibody from which the CDRs are derived to provide enhanced or different properties of interest, such as greater affinity or greater affinity A combination of avidity.
可操纵和改变抗体恒定区的某些部分，以提供具有不同与或优于亲代抗体中观察到性质的抗体同源物、衍生物、片段等。因此，例如许多IgG4抗体在铰链区附近形成链内二硫键。链内键可使亲代双价分子变得不稳定，形成含有重链及与之相连轻链的单价分子。这类分子可重新结合，但为随机结合。Certain portions of antibody constant regions can be manipulated and altered to provide antibody homologues, derivatives, fragments, etc. that have properties different from or superior to those observed in the parent antibody. Thus, for example, many IgG4 antibodies form intrachain disulfide bonds near the hinge region. Intrachain bonds can destabilize the parent bivalent molecule, forming a monovalent molecule containing a heavy chain with an associated light chain. Such molecules can reassociate, but randomly.
经观察，修饰IgG4分子铰链区的氨基酸可降低链内键形成的几率，从而稳定IgG4分子，这将最小化双特异性分子形成的几率。若治疗用抗体是IgG4分子，这种修饰则是有利的，这是因为稳定性增强可将最小化分子在生产和制备过程中以及在体内的解离几率。单价抗体可能没有与双价亲代分子相同的有效性。例如，当将双价IgG4施用患者时，双价IgG4的百分比在两周内衰变为约30％。228位的氨基酸取代增强了IgG4稳定性。位于228位的丝氨酸可为另一氨基酸替换，例如其余19种氨基酸中的一种。尤其可对重组抗体作出这种变动，其中核酸编码序列经突变可得到228位处的氨基酸替换。例如S可被替换为脯氨酸。It has been observed that modifying amino acids in the hinge region of IgG4 molecules can reduce the chance of intrachain bond formation, thereby stabilizing the IgG4 molecule, which will minimize the chance of bispecific molecule formation. This modification is advantageous if the therapeutic antibody is an IgG4 molecule, since the increased stability will minimize the chance of dissociation of the molecule during production and preparation and in vivo. Monovalent antibodies may not have the same effectiveness as the bivalent parent molecule. For example, when bivalent IgG4 is administered to a patient, the percentage of bivalent IgG4 decays to about 30% within two weeks. Amino acid substitution at position 228 enhances IgG4 stability. The serine at position 228 can be replaced by another amino acid, such as one of the remaining 19 amino acids. In particular, such changes can be made to recombinant antibodies in which the nucleic acid coding sequence has been mutated to obtain an amino acid substitution at position 228. For example S can be replaced by proline.
另一组适合修饰的氨基酸包括铰链区域的氨基酸，这些氨基酸影响含有重链的分子与Fc受体之间的结合和被结合抗体的内化。这类氨基酸包括IgG1分子中从约233至约237的残基(Glu-Leu-Leu-Gly-Gly)(SEQ ID NO:49)中；从约252至约256的残基(Met-Ile-Ser-Arg-Thr)(SEQ ID NO:50)和从约318(Glu)至约331(Pro)的残基，例如包括Lys320、Lys322和Pro329。Another group of amino acids suitable for modification includes those of the hinge region, which affect the binding of the heavy chain-containing molecule to theFc receptor and the internalization of the bound antibody. Such amino acids include residues from about 233 to about 237 in the IgG1 molecule (Glu-Leu-Leu-Gly-Gly) (SEQ ID NO: 49); residues from about 252 to about 256 (Met-Ile- Ser-Arg-Thr) (SEQ ID NO:50) and residues from about 318 (Glu) to about 331 (Pro), for example including Lys320 , Lys322 and Pro329 .
完全人抗体是人患者的治疗性治疗所特别希望的。可籍许多本领域知晓的方法制备人抗体，其中包括利用源自人免疫球蛋白序列的抗体文库而进行的上述噬菌体展示方法，参阅US专利号4,444,887和4,716,111；和WO 98/46645,WO 98/50433,WO 98/24893,WO98/16654,WO 96/34096,WO 96/33735和WO 91/10741。还可利用Cole等人和Boerder等人的技术制备人单克隆抗体(Cole等人,Monoclonal Antibodies and Cancer Therapy,AlanR.Liss(1985)；和Boerner等人,J Immunol 147:86(1991))。Fully human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be prepared by a number of methods known in the art, including the phage display method described above using antibody libraries derived from human immunoglobulin sequences, see US Patent Nos. 4,444,887 and 4,716,111; and WO 98/46645, WO 98/ 50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741. Human monoclonal antibodies can also be prepared using the techniques of Cole et al. and Boerder et al. (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss (1985); and Boerner et al., J Immunol 147:86 (1991)).
还可使用转基因小鼠生产人抗体，这些小鼠不能表达有功能的内源免疫球蛋白，但也表达某些人免疫球蛋白基因。例如，可籍随机方式或同源重组将人重链和轻链免疫球蛋白基因复合物引入到小鼠的胚胎干细胞。或者，除了人重链和轻链基因以外，可将人的可变区、恒定区和多变区引入到小鼠胚胎干细胞中。通过同源重组引入人免疫球蛋白基因座，可使小鼠重链和轻链免疫球蛋白基因分别或同时失去功能。具体而言，JH区的纯合缺失可防止内源抗体生成。被修饰的胚胎干细胞被扩展并被显微注射到胚泡中，形成嵌合小鼠。随后繁育嵌合小鼠，产生表达人抗体的纯合后代，例如参阅Jakobovitis等人,Proc NatlAcad Sci USA 90:2551(1993)；Jakobovitis等人,Nature 362:255(1993)；Bruggermann等人,Year in Immunol 7:33(1993)；和Duchosal等人,Nature 355:258(1992))。Human antibodies can also be produced using transgenic mice that do not express functional endogenous immunoglobulins, but do express certain human immunoglobulin genes. For example, human heavy and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells by random means or by homologous recombination. Alternatively, human variable, constant and multivariable regions can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The introduction of human immunoglobulin loci by homologous recombination renders the mouse heavy and light chain immunoglobulin genes individually or simultaneously nonfunctional. Specifically, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells were expanded and microinjected into blastocysts to form chimeric mice. Chimeric mice are subsequently bred to produce homozygous offspring expressing human antibodies, see, e.g., Jakobovitis et al., Proc Natl Acad Sci USA 90:2551 (1993); Jakobovitis et al., Nature 362:255 (1993); Bruggermann et al., Year in Immunol 7:33 (1993); and Duchosal et al., Nature 355:258 (1992)).
转基因小鼠按正常方式用IL-4或IL-13细胞因子如IL-4或IL-13的全部或部份进行免疫。利用常规杂交瘤技术可从免疫的转基因小鼠得到抗IL-4和IL-13的单克隆抗体。转基因小鼠所携带的人免疫球蛋白转基因在B细胞分化过程中发生重排，随后经历类别转换和体细胞突变。因此，利用这种技术有可能产生治疗用的IgG、IgA、IgM和IgE抗体。若欲了解概况，可参阅Lonberg等人,Int Rev Immunol 13:65-93(1995)。对于对生产人抗体和人单克隆抗体以及生产这类抗体的方案的讨论，可参阅例如WO 98/24893；WO 92/01047；WO 96/34096；和WO 96/33735；EPO No.0598 877；和US专利号5,413,923；5,625,126；5,633,425；5,569,825；5,661,016；5,545,806；5,814,318；5,885,793；5,916,771以及5,939,598.此外，例如Amgen(Fremont,CA)、Genpharm(San Jose,CA)和Medarex,Inc.(Princeton,NJ)等公司利用与上述类似的技术可从事抗IL-4和/或IL-13人抗体的提供。Transgenic mice are normally immunized with IL-4 or IL-13 cytokines, such as all or part of IL-4 or IL-13. Monoclonal antibodies against IL-4 and IL-13 can be obtained from immunized transgenic mice using conventional hybridoma technology. Transgenic mice harbor human immunoglobulin transgenes that rearrange during B-cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using this technique it is possible to generate therapeutic IgG, IgA, IgM and IgE antibodies. For an overview see Lonberg et al., Int Rev Immunol 13:65-93 (1995). For a discussion of the production of human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., WO 98/24893; WO 92/01047; WO 96/34096; and WO 96/33735; EPO No. 0598 877; and US Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; ) and other companies can engage in the provision of anti-IL-4 and/or IL-13 human antibodies using techniques similar to those described above.
同样，可免疫移植有人外周血白细胞、脾细胞或骨髓(例如以色列XTLBiopharmaceuticals公司的三源杂交瘤技术)的小鼠，制备人mAbs。可利用被称为"导向选择"的技术生成识别所选择表位的完全人抗体。在该方法中，使用经选择的非人单克隆抗体如小鼠抗体来引导识别相同表位的完全人抗体的选择(Jespers等人,Bio/technology 12:899(1988))。Similarly, human mAbs can be prepared by immunizing mice transplanted with human peripheral blood leukocytes, splenocytes or bone marrow (for example, the triple hybridoma technology of XTL Biopharmaceuticals, Israel). Fully human antibodies that recognize selected epitopes can be generated using a technique known as "guided selection." In this method, selected non-human monoclonal antibodies, such as mouse antibodies, are used to guide the selection of fully human antibodies that recognize the same epitope (Jespers et al., Bio/technology 12:899 (1988)).
使用重组技术时，抗体变异体可在细胞内、周质空间内产生，或直接被分泌到培养基中。如果抗体变异体是在细胞内产生的，则第一步，例如通过离心或超滤除去微小碎片，其或是宿主细胞或是被裂解的片段。Carter等人,Bio/Technology 10:163(1992)描述了一种分离被分泌到大肠杆菌周质空间的抗体的方法。简而言之，细胞浆与乙酸钠(pH 3.5)和EDTA接触。可通过离心除去细胞碎片。当抗体变异体被分泌到培养基中时，一般首先利用市售蛋白质浓缩过滤器如Amicon或Millipore Pellicon的超滤组件浓缩来自这些表达体系的上清液。可加入蛋白酶抑制剂例如PMSF以抑制蛋白酶解，还可加入抗生素以防止外来污染物的生长。Using recombinant techniques, antibody variants can be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody variant is produced intracellularly, the first step, eg, by centrifugation or ultrafiltration, removes minute debris, either host cells or lysed fragments. Carter et al., Bio/Technology 10:163 (1992) describe a method for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, cell plasma was contacted with sodium acetate (pH 3.5) and EDTA. Cellular debris can be removed by centrifugation. When antibody variants are secreted into culture medium, supernatants from these expression systems are typically first concentrated using commercially available protein concentration filters such as ultrafiltration modules from Amicon or Millipore Pellicon. Protease inhibitors such as PMSF can be added to inhibit proteolysis, and antibiotics can also be added to prevent the growth of adventitious contaminants.
可使用例如羟磷灰石层析、凝胶电泳、透析和亲和层析纯化从细胞制备的抗体组合物。蛋白质A或蛋白质G是否适合作为亲和配体取决于抗体变异体上任意免疫球蛋白Fc结构域的种类和同种型。可使用蛋白质A纯化基于人IgG1、IgG2或IgG4重链的抗体(Lindmark等人,J Immunol Meth 62:1(1983))。可使用蛋白质G用于小鼠同种型和人IgG3(Guss等人,EMBO J 5:1567(1986))。亲和配体所结合的基质最常见是琼脂糖，但也可使用其它基质。机械稳定的基质例如可控孔玻璃或聚(苯乙烯二乙烯)苯可允许比琼脂糖所实现的更快的流速和更短的处理时间。当抗体变异体包括CH3结构域时，可使用Bakerbond ABXTM树脂(JTBaker；Phillipsburg,NJ)进行纯化。还可利用其它蛋白质纯化技术，例如离子交换柱分级分离、乙醇沉淀、反相HPLC、二氧化硅上的层析、肝素琼脂糖上的层析、阴离子或阳离子交换树脂上的层析(例如多聚天冬氨酸柱)、色谱聚焦、SDS-PAGE和硫酸铵沉淀，这取决于待回收的抗体或变异体。Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography. Whether protein A or protein G is suitable as an affinity ligand depends on the class and isotype of any immunoglobulinFc domain on the antibody variant. Antibodies based on human IgGl, IgG2, or IgG4 heavy chains can be purified using Protein A (Lindmark et al., J Immunol Meth 62:1 (1983)). Protein G can be used for mouse isotypes and human IgG3 (Guss et al., EMBO J 5:1567 (1986)). The matrix to which the affinity ligand is bound is most commonly agarose, but other matrices may also be used. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene may allow faster flow rates and shorter processing times than achieved with agarose. When the antibody variant includes aCH3 domain, Bakerbond ABX™ resin (JT Baker; Phillipsburg, NJ) can be used for purification. Other protein purification techniques, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins (e.g. poly polyaspartic acid column), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation, depending on the antibody or variant to be recovered.
进行任何初步纯化步骤后，含有目的抗体或目的变异体和污染物的混合物可进行低pH疏水相互作用层析，其中使用pH约2.5-4.5的洗脱缓冲液，优选在低盐浓度(例如约0-0.25M盐)下进行。Following any preliminary purification steps, the mixture containing the antibody or variant of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).
本发明的抗体可为双特异性抗体。双特异性抗体可为单克隆抗体，优选为具有至少两种不同抗原结合特异性的人或人源化抗体。在一个优选的实施方案中，双特异抗体、其片段等等对IL-4和IL-13具有结合特异性。Antibodies of the invention may be bispecific antibodies. Bispecific antibodies may be monoclonal antibodies, preferably human or humanized antibodies, having at least two different antigen-binding specificities. In a preferred embodiment, the diabodies, fragments thereof, etc. have binding specificities for IL-4 and IL-13.
生产双特异性抗体的方法是众所周知的。传统上，双特异性抗体的重组生产是基于两个免疫球蛋白重链/轻链对的共表达，其中两条重链具有不同的特异性(Milstein等人,Nature 305:537(1983))。由于免疫球蛋白重链和轻链的随机分配，杂交瘤(细胞杂交瘤(quadroma))产生10种不同抗体分子的潜在混合物，其中仅有一种具有正确的双特异性结构。通常通过亲和层析步骤进行正确分子的纯化。类似方法披露于WO 93/08829和Traunecker等人,EMBO J 10:3655(1991)。例如Kufer等人,Trends Biotech 22:238-244,2004提供了生产双特异性抗体的其它方法。Methods for producing bispecific antibodies are well known. Traditionally, recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain/light chain pairs, where the two heavy chains have different specificities (Milstein et al., Nature 305:537 (1983)) . Due to the random assortment of immunoglobulin heavy and light chains, hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is usually performed by an affinity chromatography step. Similar methods are disclosed in WO 93/08829 and Traunecker et al., EMBO J 10:3655 (1991). For example, Kufer et al., Trends Biotech 22:238-244, 2004 provide other methods for producing bispecific antibodies.
可将具有所需结合特异性的抗体可变结构域融合到免疫球蛋白的恒定结构域序列上。优选yu包括至少部分铰链区、CH2和CH3区的免疫球蛋白重链恒定结构域进行融合。它可具有首个重链恒定区(CH1)，其含有至少在一个融合中出现的轻链结合所需的位点。将编码免疫球蛋白重链融合区和免疫球蛋白轻链(若需要)的DNAs插入到不同的表达载体，并共转化到适宜的宿主生物体内。对于产生双特异性抗体的更多细节，可参阅例如Suresh等人,Meth Enzym 121:210(1986)。Antibody variable domains with the desired binding specificities can be fused to immunoglobulin constant domain sequences. Preferably the fusion is with an immunoglobulin heavy chain constant domain comprising at least part of the hinge,CH2 andCH3 regions. It may have a first heavy chain constant region (CH1 ) containing the site required for light chain binding present in at least one fusion. DNAs encoding the immunoglobulin heavy chain fusion region and the immunoglobulin light chain (if desired) are inserted into separate expression vectors and cotransformed into a suitable host organism. For more details on the generation of bispecific antibodies see, eg, Suresh et al., Meth Enzym 121:210 (1986).
本发明也考虑了异缀合物抗体。异缀合物抗体由两个共价键连接的抗体组成。已经建议例如用这类抗体靶向免疫系统细胞至不想要的细胞(US专利号4,676,980)。考虑，该抗体可用合成蛋白化学的已知方法在体外制备，包括涉及交联剂的那些方法。例如，免疫毒素可用二硫化物交换反应或形成硫酯键的方式来构建。适合于该目的之试剂的例子包括iminothiolate)和甲基-4-mercaptobutyrimidate，以及如US专利号4,676,980中所披露的那些。Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies consist of two covalently linked antibodies. It has been suggested, for example, to use such antibodies to target immune system cells to unwanted cells (US Patent No. 4,676,980). It is contemplated that the antibodies can be prepared in vitro by known methods of synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins can be constructed by disulfide exchange reactions or by the formation of thioester bonds. Examples of reagents suitable for this purpose include iminothiolate) and methyl-4-mercaptobutyrimidate, and those disclosed in US Patent No. 4,676,980.
此外，可以产生IL-4和/或IL-13的单结构域抗体。这种技术的某些例子，已在WO9425591谈及源自骆驼科重链Ig的抗体时有所叙述，以及在US20030130496谈及从噬菌体文库分离单结构域全人抗体时也有所叙述。In addition, single domain antibodies to IL-4 and/or IL-13 can be raised. Some examples of this technique are described in WO9425591 for antibodies derived from heavy chain Igs of Camelidae, and in US20030130496 for isolation of single domain fully human antibodies from phage libraries.
可选地，某些描述用于生产单链抗体的技术(US专利号4,946,778；Bird,Science242:423(1988)；Huston等人,Proc Natl Acad Sci USA 85:5879(1988)；以及Ward,等人,Nature 334:544(1989))也可适应于生产单链抗体。单链抗体是通过将Fv域的重链和轻链片段以氨基酸桥连接生成单链多肽而形成的。大肠杆菌中的功能Fv片段的组装技术也可以利用(Skerra等人,Science 242:1038(1988))。Alternatively, certain techniques are described for the production of single chain antibodies (US Pat. No. 4,946,778; Bird, Science 242:423 (1988); Huston et al., Proc Natl Acad Sci USA 85:5879 (1988); and Ward, et al. Human, Nature 334:544 (1989)) can also be adapted for the production of single chain antibodies. Single-chain antibodies are formed by linking the heavy and light chain fragments of theFv domain with an amino acid bridge to generate a single-chain polypeptide. Assembly techniques for functionalFv fragments in E. coli are also available (Skerra et al., Science 242:1038 (1988)).
本发明包括了以重组方式与多肽融合或化学缀合(包括共价和非共价缀合)的抗体。本发明的融合或辍合的抗体可用于简化纯化，参阅例如WO 93/21232；EP 439,095；Naramura等人,Immunol Lett 39:91(1994)；US专利号5,474,981；Gillies等人,Proc NatlAcad Sci USA 89:1428(1992)；以及Fell等人,J Immunol 146:2446(1991)。标记氨基酸序列可以是六组氨酸肽，如pQE载体提供的标签(QIAGEN,Inc.,Chatsworth,CA)，此外，其中许多还可经商业渠道获得，Gentz等人,Proc Natl Acad Sci USA 86:821(1989)。其它可用于纯化的肽标签包括但不限于"HA"标签，它对应于一个源自流感血凝素蛋白的表位(Wilson等人,Cell 37:767(1984))以及"flag"标签。The invention includes antibodies recombinantly fused or chemically conjugated (including covalently and non-covalently) to a polypeptide. The fused or conjugated antibodies of the invention can be used to simplify purification, see e.g. WO 93/21232; EP 439,095; Naramura et al., Immunol Lett 39:91 (1994); US Patent No. 5,474,981; Gillies et al., Proc Natl Acad Sci USA 89:1428 (1992); and Fell et al., J Immunol 146:2446 (1991). The marker amino acid sequence can be a hexahistidine peptide, such as the tag provided by the pQE vector (QIAGEN, Inc., Chatsworth, CA), many of which are also commercially available, Gentz et al., Proc Natl Acad Sci USA 86: 821 (1989). Other peptide tags that can be used for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984)), and the "flag" tag.
还可创造单肽链结合分子，其中重链和轻链Fv区域相连。单链抗体("scFv")和它们的构建方法在例如US专利号4,946,778中有所叙述。或者，Fab可以类似方式构建和表达。与完全的鼠类单克隆抗体相比，所有完全和部分的人抗体均可具有较小的免疫原性，片段和单链抗体也可具有较小的免疫原性。Single peptide chain binding molecules can also be created in which the heavy and light chainFv regions are linked. Single-chain antibodies ("scFv") and methods for their construction are described, eg, in US Patent No.4,946,778 . Alternatively, Fab can be constructed and expressed in a similar manner. All fully and partially human antibodies can be less immunogenic than fully murine monoclonal antibodies, as can fragments and single-chain antibodies.
抗体或抗体片段，可从用McCafferty等人,Nature 348:552(1990)所述技术产生的抗体噬菌体文库分离。Clarkson等人,Nature 352:624(1991)和Marks等人,J Mol Biol222:581(1991)分别叙述了使用噬菌体文库分离鼠类和人抗体。随后的出版物叙述了以链改组生产高亲和力(在nM的范围内)人抗体(Marks等人,Bio/Technology 10:779(1992))，以及以组合感染和体内重组作为构建很大噬菌体文库的策略(Waterhouse等人,NuclAcids Res 21:2265(1993))。因此，这些技术是分离单克隆抗体的传统单克隆抗体杂交瘤技术的可行替代。Antibodies, or antibody fragments, can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552 (1990). Clarkson et al., Nature 352:624 (1991) and Marks et al., J Mol Biol 222:581 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high-affinity (in the nM range) human antibodies by chain shuffling (Marks et al., Bio/Technology 10:779 (1992)), as well as the construction of very large phage libraries by combinatorial infection and in vivo recombination. strategy (Waterhouse et al., Nucl Acids Res 21:2265 (1993)). Therefore, these techniques are viable alternatives to traditional mAb hybridoma techniques for isolation of mAbs.
候选的抗IL-4和/或IL-13抗体是用酶联免疫吸附测定(ELISA)、FACS、Western免疫印迹技术或本技术领域内已知的其它免疫化学技术检测的。Candidate anti-IL-4 and/or IL-13 antibodies are detected using enzyme-linked immunosorbent assay (ELISA), FACS, Western immunoblotting techniques or other immunochemical techniques known in the art.
为了确定特定的抗体同源物是否与人IL-4和/或IL-13结合，任何传统的结合测定均可应用。有用的IL-4和IL-13结合测定包括FACS分析、ELISA测定、表面等离振子共振(Biacore)、放射免疫分析等等，这些方法检测抗体与人IL-4和/或IL-13的结合以及由此产生的功能。本文所教导的全长和可溶形式的人IL-4和IL-13在这类测定中是有用的。抗体或同源物与IL-4和/或IL-13或其可溶性片段的结合，可很方便地通过使用对该物种的免疫球蛋白具有特异性的第二种抗体来检测，所检测的抗体或同源物源自上述物种。To determine whether a particular antibody homologue binds to human IL-4 and/or IL-13, any conventional binding assay may be used. Useful IL-4 and IL-13 binding assays include FACS analysis, ELISA assay, surface plasmon resonance (Biacore), radioimmunoassay, etc., which detect binding of antibodies to human IL-4 and/or IL-13 and the resulting functions. Full length and soluble forms of human IL-4 and IL-13 as taught herein are useful in such assays. Binding of the antibody or homologue to IL-4 and/or IL-13, or soluble fragments thereof, is conveniently detected by use of a second antibody specific for an immunoglobulin of that species, the detected antibody Or homologues derived from the above species.
为了确定特定的抗体或同源物是否显著地阻断IL-4和/或IL-13的结合，任何适宜的竞争测定均可使用。有用的测定包括例如ELISA测定、FACS测定、放射免疫测定等，它们定量抗体或同源物与IL-4和/或IL-13结合的竞争能力。优选地，配体阻断标记的人IL-4和/或IL-13与固定化抗体或同源物结合的能力得以测量。To determine whether a particular antibody or homologue significantly blocks IL-4 and/or IL-13 binding, any suitable competition assay may be used. Useful assays include, eg, ELISA assays, FACS assays, radioimmunoassays, and the like, which quantify the ability of an antibody or homologue to compete for IL-4 and/or IL-13 binding. Preferably, the ability of the ligand to block the binding of labeled human IL-4 and/or IL-13 to the immobilized antibody or homologue is measured.
本发明之抗体可在抗体识别或特异性结合的IL-4和/或IL-13的表位或部分方面来描述或规定。该表位或多肽部分可按本文所述而规定，例如通过N端和C端的位置、通过邻近氨基酸残基的大小、构象表位等。Antibodies of the invention may be described or specified in terms of the epitope or portion of IL-4 and/or IL-13 that the antibody recognizes or specifically binds to. The epitope or polypeptide portion may be defined as described herein, eg, by N-terminal and C-terminal position, by size of adjacent amino acid residues, conformational epitope, and the like.
本发明之抗体也可在交叉反应性方面来描述或规定。结合与IL-4和/或IL-13有至少95％、至少90％、至少85％、至少80％、至少75％、至少70％、至少有65％、至少有60％、至少55％，以及至少50％同一性(使用本技术领域内已知和本文所述的方法计算)的IL-4和/或IL-13多肽的抗体，也包括在本发明内。Antibodies of the invention may also be described or specified in terms of cross-reactivity. binds to IL-4 and/or IL-13 at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, Also included in the invention are antibodies to IL-4 and/or IL-13 polypeptides that are at least 50% identical (calculated using methods known in the art and described herein).
本发明之抗体也可以在与IL-4和/或IL-13的结合亲和力方面来描述或规定。抗IL-4和/或IL-13抗体可以低于约10-7M、低于约10-6M，或低于约10-5M的KD结合。目的抗体的较高结合亲和力可以是有益的，例如具有约10-8至约10-15M、约10-8至约10-12M、约10-9至约10-11M，或约10-8至约10-10M平衡解离常数或KD的那些抗体。本发明还提供了一些抗体，如本技术领域内任何已知的测定竞争性结合的方法如本文所述的免疫测定所确定，这些抗体竞争性地抑制抗体与本发明之表位的结合。在一些优选的实施方案中，抗体竞争性地抑制与表位的结合达至少95％、至少90％、至少85％、至少80％、至少75％、至少70％、至少为60％，或至少50％。Antibodies of the invention may also be described or specified in terms of binding affinity to IL-4 and/or IL-13. Anti-IL-4 and/or IL-13 antibodies may bind with aKD of less than about 10"7M , less than about 10"6M , or less than about 10"5M . A higher binding affinity of the antibody of interest may be beneficial, for example having about 10"8 to about 10"15 M, about 10"8 to about 10"12 M, about 10"9 to about 10"11 M, or about 10 Those antibodies having an equilibrium dissociation constant orKD of-8 to about 10-10 M. The invention also provides antibodies that competitively inhibit binding of antibodies to epitopes of the invention, as determined by any method known in the art for determining competitive binding, such as the immunoassays described herein. In some preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
本发明还包括含有目的抗体的辍合物。该辍合物包含两个主要组分，目的抗体和可以是一种细胞结合剂、细胞毒性剂等的第二个组分。The invention also includes conjugates comprising the antibody of interest. The conjugate comprises two main components, the antibody of interest and a second component which may be a cell-binding agent, cytotoxic agent, or the like.
本文所用的术语"细胞结合剂"是指一种能特异性识别细胞表面的分子并与其结合的活性剂。因此，细胞结合剂可以是CD抗原、病原体抗原如病毒抗原，分化抗原，癌症抗原，细胞特异性抗原，组织特异性抗原，Ig或Ig样分子等。The term "cell-binding agent" as used herein refers to an active agent that can specifically recognize and bind to a molecule on the surface of a cell. Thus, the cell-binding agent may be a CD antigen, a pathogen antigen such as a viral antigen, a differentiation antigen, a cancer antigen, a cell-specific antigen, a tissue-specific antigen, an Ig or an Ig-like molecule, and the like.
细胞结合剂可以是目前已知或刚为人所知的任何类型，包括肽、非肽、糖类、核酸、配体、受体等，或其组合。该细胞结合剂可以是能与细胞结合的任何化合物，无论是以特异性或非特异性方式结合。一般而言，细胞结合剂可以是抗体(尤其是单克隆抗体)、淋巴因子、激素、生长因子、维生素，营养输送分子(如转铁蛋白)，或其它任何细胞结合分子或物质。Cell-binding agents can be of any type currently known or just now known, including peptides, non-peptides, carbohydrates, nucleic acids, ligands, receptors, etc., or combinations thereof. The cell-binding agent can be any compound capable of binding to cells, whether in a specific or non-specific manner. In general, a cell-binding agent can be an antibody (especially a monoclonal antibody), a lymphokine, a hormone, a growth factor, a vitamin, a nutrient transport molecule (such as transferrin), or any other cell-binding molecule or substance.
可利用的细胞结合剂的其它例子包括：多克隆抗体；单克隆抗体；和抗体片段，如Fab、Fab'、F(ab')2以及Fv(Parham,J.Immunol.131:2895-2902(1983)；Spring等人,J.Immunol.113:470-478(1974)；以及Nisonoff等人,Arch.Biochem.Biophys.89：230-244(1960))。Other examples of cell-binding agents that may be used include: polyclonal antibodies; monoclonal antibodies; and antibody fragments such as Fab,Fab' , F(ab')2 , andFv (Parham, J. Immunol. 131:2895 -2902 (1983); Spring et al., J. Immunol. 113:470-478 (1974); and Nisonoff et al., Arch. Biochem. Biophys. 89:230-244 (1960)).
第二个组分也可以是一种细胞毒性剂。本文所用的术语"细胞毒性剂"是指能降低或阻断细胞的功能或生长，和/或导致细胞破坏的物质。因此，细胞毒性剂可以是一种紫杉醇、美登素(maytansinoids)，如DM1或DM4、CC-1065或CC－1065类似物、蓖麻毒蛋白、丝裂霉素C等。在某些实施方案中，如同本发明之辍合物的任何结合剂，细胞毒性剂是以共价键与目的抗体直接或通过一个可切割的或不可切割的连接体连接的。The second component can also be a cytotoxic agent. As used herein, the term "cytotoxic agent" refers to a substance that reduces or blocks the function or growth of cells, and/or causes the destruction of cells. Thus, the cytotoxic agent may be a paclitaxel, maytansinoids such as DM1 or DM4, CC-1065 or CC-1065 analogs, ricin, mitomycin C, and the like. In certain embodiments, as with any binding agent of the conjugates of the invention, the cytotoxic agent is covalently linked to the antibody of interest either directly or through a cleavable or non-cleavable linker.
适宜的美登素的例子包括美登醇(maytansinol)和美登醇类似物。美登素能抑制微管的形成并对哺乳动物细胞具有很强的毒性。Examples of suitable maytansinols include maytansinol and maytansinol analogs. Maytansine inhibits microtubule formation and is highly toxic to mammalian cells.
适宜的美登醇类似物的例子包括含有经修饰芳环的那些和在其它位置有修饰的那些美登素醇类似物。在下列US专利中披露了这类适宜的美登素：4,424,219；4,256,746；4,294,757；4,307,016；4,313,946；4,315,929；4,331,598；4,361,650；4,362,663；4,364,866；4,450,254；4,322,348；4,371,533；6,333,410；5,475,092；5,585,499；以及5,846,545。Examples of suitable maytansinol analogs include those containing modified aromatic rings and those with modifications at other positions.在下列US专利中披露了这类适宜的美登素：4,424,219；4,256,746；4,294,757；4,307,016；4,313,946；4,315,929；4,331,598；4,361,650；4,362,663；4,364,866；4,450,254；4,322,348；4,371,533；6,333,410；5,475,092；5,585,499；以及5,846,545。
适宜的含有一个经修饰芳环的美登醇类似物的例子包括：(1)C-19-脱氯(US专利号4,256,746)(例如通过ansamytocin P2的LAH还原而制备)；(2)C-20-羟基(或C-20-脱甲基)+/-C-19-脱氯(US专利号4,361,650和4,307,016)(例如用链霉菌属(Stretomyces)或放线菌属(Actinomyces)脱甲基或用氢化锂铝(LAH)脱氯而制备)；以及(3)C-20-脱甲氧基、C-20-酰氧基(-OCOR)，+/-脱氯(US专利号4,294,757)(用酰基氯酰化而制备)。Examples of suitable maytansinol analogs containing a modified aromatic ring include: (1) C-19-dechlorination (US Patent No. 4,256,746) (prepared, for example, by LAH reduction of ansamytocin P2); (2) C- 20-Hydroxy (or C-20-demethylation) +/- C-19-dechlorination (US Patent Nos. 4,361,650 and 4,307,016) (e.g. demethylation with Stretomyces or Actinomyces or prepared by dechlorination of lithium aluminum hydride (LAH); and (3) C-20-demethoxy, C-20-acyloxy (-OCOR), +/-dechlorination (US Patent No. 4,294,757) (Prepared by acylation with acid chloride).
适宜的在其它位置有修饰的美登醇类似物的例子包括：(1)C-9-SH(US专利号4,424,219)(通过美登醇与H2S或P2S5的反应而制备)；(2)C-14烷氧基甲基(脱甲氧基/CH2OR)(US专利号4,331,598)；(3)C-14-羟甲基或酰基氧基甲基(CH2OH或CH2OAc)(US专利号4,450,254)(从诺卡氏菌属(Nocardia)制备)；(4)C-15-羟基/酰基氧基(US专利号4,364,866)(通过链霉菌使美登醇转化而制备)；(5)C-15-甲氧基(US4,313,946和4,315,929)(从Trewianudiflora分离)；(6)C-18-N-脱甲基(US专利号4,362,663和4,322,348)(通过链霉菌使美登醇脱甲基化而制备)；以及(7)4,5-脱氧基(US专利号4,371,533)(通过美登醇的三氯化钛/LAH还原而制备)。Examples of suitable maytansinol analogs with modifications at other positions include: (1) C-9 -SH (US PatentNo.4,424,219 ) (prepared by reaction of maytansinol with H2S or P2S5) ; (2) C-14 alkoxymethyl (demethoxy/CH2 OR) (US Patent No. 4,331,598); (3) C-14-hydroxymethyl or acyloxymethyl (CH2 OH or CH2OAc) (US PatentNo. 4,450,254) (prepared from Nocardia); (4) C-15-hydroxy/acyloxy (US Patent No. 4,364,866) (conversion of maytansinol by Streptomyces (5) C-15-methoxy (US 4,313,946 and 4,315,929) (isolated from Trewianudiflora); (6) C-18-N-demethylation (US Pat. Nos. 4,362,663 and 4,322,348) (via chain mold demethylation of maytansinol); and (7) 4,5-deoxyl (US Patent No. 4,371,533) (prepared by titanium trichloride/LAH reduction of maytansinol).
细胞毒性辍合物可以体外方法制备。为了将细胞毒性剂、药物或前药与抗体连接，通常使用一个连接基。适宜的连接基是本技术领域内周知的，包括二硫基、硫醚基、酸不稳定基、光不稳定基(photolabile group)，肽酶不稳定基以及酯酶不稳定基。例如，利用二硫化物交换反应，或在目的抗体和药物或前体药之间形成硫醚键，即可构建辍合物。Cytotoxic conjugates can be prepared by in vitro methods. To attach a cytotoxic agent, drug or prodrug to an antibody, a linker is typically used. Suitable linking groups are well known in the art and include disulfide groups, thioether groups, acid labile groups, photolabile groups, peptidase labile groups and esterase labile groups. For example, conjugates can be constructed using disulfide exchange reactions, or the formation of thioether linkages between the antibody of interest and a drug or prodrug.
如上所讨论，本发明提供了编码本文所披露抗体或其功能片段或变体变体的分离的核酸序列，含有编码本发明的抗体或其功能片段的IL-4和/或IL-13结合部分的核苷酸序列的载体构建体，含有这种载体的宿主细胞，以及生产多肽的重组技术。As discussed above, the invention provides isolated nucleic acid sequences encoding the antibodies or functional fragments or variant variants thereof disclosed herein, comprising an IL-4 and/or IL-13 binding portion encoding an antibody or functional fragment thereof of the invention vector constructs of the nucleotide sequence, host cells containing such vectors, and recombinant techniques for producing polypeptides.
该载体通常包含本技术领域内已知的组分，且一般包括但不限于一个或多个下列：信号序列、复制起点，一个或多个标记或选择基因，促进和/或增强翻译的序列，增强子元件等。因此，表达载体包括与适宜的转录或转译调控核苷酸序列，例如那些源自哺乳动物、微生物、病毒或昆虫基因的核苷酸序列，有效地连接的核苷酸序列。其它调控序列的例子包括操纵子、mRNA核糖体结合位点，和/或其它控制转录和翻译(例如其起始和终止)的适宜序列。当调控序列与适当多肽的核苷酸序列功能相关时，核苷酸序列就是"有效连接的"。因此，如果启动子核苷酸序列控制该核苷酸序列的转录，那么该启动子核苷酸序列就是与例如抗体重链序列有效连接。The vector generally comprises components known in the art and generally includes, but is not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker or selection genes, sequences that promote and/or enhance translation, Enhancer elements, etc. Thus, expression vectors include nucleotide sequences operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from mammalian, microbial, viral or insect genes. Examples of other regulatory sequences include operators, mRNA ribosomal binding sites, and/or other suitable sequences that control transcription and translation (eg, initiation and termination thereof). Nucleotide sequences are "operably linked" when the regulatory sequence is functionally related to the nucleotide sequence of the appropriate polypeptide. Thus, a promoter nucleotide sequence is operably linked to, for example, an antibody heavy chain sequence if the promoter nucleotide sequence controls the transcription of that nucleotide sequence.
此外，编码与抗体重链和/或轻链序列非天然相连的适当信号肽的序列，可纳入表达载体。例如，信号肽(分泌前导)的核苷酸序列可与多肽序列符合读框地融合，使得该抗体被分泌到周质空间或培养基中。在想要的宿主细胞中起作用的信号肽，增强了适当抗体或其部分的细胞外分泌。该信号肽可在细胞分泌抗体时从多肽上被切割下来。这类分泌信号的例子是众所周知的并包括例如在US专利号5,698,435、5,698,417，以及6,204,023中所述的那些分泌信号。In addition, sequences encoding appropriate signal peptides non-naturally associated with antibody heavy and/or light chain sequences may be incorporated into expression vectors. For example, a nucleotide sequence for a signal peptide (secretion leader) can be fused in-frame to a polypeptide sequence such that the antibody is secreted into the periplasmic space or into the culture medium. A signal peptide acting in the desired host cell enhances the extracellular secretion of the appropriate antibody or portion thereof. The signal peptide can be cleaved from the polypeptide when the cell secretes the antibody. Examples of such secretion signals are well known and include, for example, those described in US Pat. Nos. 5,698,435, 5,698,417, and 6,204,023.
载体可以是质粒、单链或双链病毒载体、单链或双链RNA或DNA噬菌体载体、噬菌粒、黏粒或任何其它目的转基因的载体。采用众所周知的将DNA和RNA导入细胞的技术，可将此类载体作为多核苷酸导入细胞。在噬菌体和病毒载体的情况下，也可采用众所周知的感染及转导技术，将载体作为包装或包囊的病毒导入细胞。病毒载体可以是可复制型或复制缺陷型。在后一种情况下，病毒的繁殖一般只会在补充型(complementing)宿主细胞中发生，并需使用载有产生颗粒所必须的多种病毒成分的多种载体。使用源自现有DNA构建体的RNA，无细胞翻译系统也可用于生产蛋白(参阅例如WO 86/05807和WO 89/01036，以及US专利号5,122,464)。The vector can be a plasmid, a single-stranded or double-stranded viral vector, a single-stranded or double-stranded RNA or DNA phage vector, a phagemid, a cosmid or any other target transgenic vector. Such vectors can be introduced into cells as polynucleotides using well known techniques for introducing DNA and RNA into cells. In the case of phage and viral vectors, the vectors can also be introduced into cells as packaged or encapsulated viruses using well known infection and transduction techniques. Viral vectors can be replication competent or replication defective. In the latter case, virus propagation generally only occurs in complementing host cells, and requires the use of multiple vectors carrying the various viral components necessary for particle production. Cell-free translation systems can also be used to produce proteins using RNA derived from existing DNA constructs (see eg WO 86/05807 and WO 89/01036, and US Patent No. 5,122,464).
本发明之抗体可从任何适宜的宿主细胞表达。可用于本发明的宿主细胞的例子包括原核细胞、酵母或较高级的真核细胞，并包括但不限于微生物如用含有目的抗体编码序列的重组噬菌体DNA、质粒DNA或黏粒DNA表达载体转化的细菌(如大肠杆菌、枯草芽孢杆菌(B.subtilis)、肠杆菌属(Enterobacter)，欧文氏菌属(Erwinia)，克雷伯菌属(Klebsiella)，变形杆菌属(Proteus)、沙门氏菌属(Salmonella)、沙雷菌属(Serratia)和志贺菌属(Shigella)，以及杆菌(Bacilli)、假单胞菌属(Pseudomonas)和链霉菌属)；用含有抗体编码序列的重组酵母表达载体转化的酵母(例如酵母属(Saccharomyces)、毕赤酵母属(Pichia)、放线菌(Actinomycetes)、克鲁维酵母属(Kluyveromyces)、裂殖酵母属(Schizosaccharomyces)、念珠菌属(Candida)、木霉属(Trichoderma)，链孢霉属(Neurospora)，以及丝状真菌，如链孢霉属(Neurospora)、青霉属(Penicillium,)、Tolypocladium和曲霉属(Aspergillus))；用含有抗体编码序列的重组病毒表达载体(如杆状病毒)感染的昆虫细胞系统；用重组病毒表达载体(如花椰菜花叶病毒、CaMV；或烟草花叶病毒，TMV)感染的，或用含有抗体编码序列的重组质粒表达载体(例如Ti质粒)转化的植物细胞系统；或包含重组表达构建体的哺乳动物细胞系统(例如COS、CHO、BHK、293或3T3细胞)。该构建体含有源自哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或源自哺乳动物病毒的启动子(例如腺病毒后期启动子；或痘苗病毒7.5K启动子)。Antibodies of the invention can be expressed from any suitable host cell. Examples of host cells that can be used in the present invention include prokaryotic cells, yeast, or higher eukaryotic cells, and include, but are not limited to, microorganisms such as those transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequence of interest. Bacteria (such as Escherichia coli, B.subtilis, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella ), Serratia, and Shigella, and Bacilli, Pseudomonas, and Streptomyces); yeast transformed with a recombinant yeast expression vector containing the antibody coding sequence (e.g. Saccharomyces, Pichia, Actinomycetes, Kluyveromyces, Schizosaccharomyces, Candida, Trichoderma (Trichoderma, Neurospora, and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus); Insect cell systems infected with viral expression vectors (eg, baculovirus); infected with recombinant viral expression vectors (eg, cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV), or expressed from recombinant plasmids containing antibody coding sequences A plant cell system transformed with a vector (eg Ti plasmid); or a mammalian cell system (eg COS, CHO, BHK, 293 or 3T3 cells) comprising a recombinant expression construct. The construct contains a promoter derived from the genome of a mammalian cell (eg, the metallothionein promoter) or a promoter derived from a mammalian virus (eg, the adenovirus late promoter; or the vaccinia virus 7.5K promoter).
用于原核宿主细胞的表达载体，一般含有一个或多个表型选择标记基因。表型选择标记基因是，例如编码赋予抗生素抗性或提供自养要求的蛋白的基因。有用的原核宿主细胞的表达载体的例子包括从商业可获得的质粒如pKK223-3(Pharmacia FineChemicals,Uppsala,Sweden)、pGEM1(Promega Biotec,Madison,WI)、pET(Novagen,Madison,WI)以及pRSET(Invitrogen,Carlsbad,CA)系列载体(Studier,J Mol Biol 219:37(1991)；以及Schoepfer,Gene 124:83(1993))所来源的那些载体。通常用于重组原核宿主细胞表达载体的启动子序列包括T7，(Rosenberg等人,Gene 56:125(1987))、β-内酰胺酶(青霉素酶)、乳糖启动子系统(Chang等人,Nature 275:615(1978)；以及Goeddel等人,Nature 281:544(1979))、色氨酸(trp)启动子系统(Goeddel等人,Nucl Acids Res 8:4057(1980))，以及tac启动子(Sambrook等人,Molecular Cloning,A Laboratory Manual,2版，Cold Spring Harbor Laboratory(1990))。Expression vectors for prokaryotic host cells generally contain one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or provides for an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include commercially available plasmids such as pKK223-3 (Pharmacia FineChemicals, Uppsala, Sweden), pGEM1 (Promega Biotec, Madison, WI), pET (Novagen, Madison, WI), and pRSET (Invitrogen, Carlsbad, CA) series of vectors (Studier, J Mol Biol 219:37 (1991); and Schoepfer, Gene 124:83 (1993)). Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include T7, (Rosenberg et al., Gene 56:125 (1987)), β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615 (1978); and Goeddel et al., Nature 281:544 (1979)), the tryptophan (trp) promoter system (Goeddel et al., Nucl Acids Res 8:4057 (1980)), and the tac promoter (Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (1990)).
酵母载体往往含有复制起点序列，如来自2μ酵母质粒的、自主复制序列(ARS)、启动子区域、多聚腺苷化序列、转录终止序列以及可选择标记基因。酵母载体的适宜的启动子序列包括下述的启动子：金属硫蛋白、3-磷酸甘油酸酯激酶(Hitzeman等人,J Biol Chem255:2073(1980))或其它糖酵解酶(Holland等人,Biochem 17:4900(1978))如烯醇化酶、甘油醛-3-磷酸脱氢酶、己糖激酶、丙酮酸脱羧酶、果糖磷酸激酶、葡糖-6-磷酸异构酶、3-磷酸甘油酸酯变位酶、丙酮酸激酶、丙糖磷酸异构酶、磷酸葡糖异构酶以及葡糖激酶，等等。其它用于酵母表达的适宜载体和启动子，在Fleer等人,Gene 107:285(1991)中进一步说明。其它酵母和酵母转化方案的适宜启动子和载体是本技术领域内众所周知的。酵母转化方案也是众所周知的。Hinnen等人,Proc Natl Acad Sci 75:1929(1978)叙述了这样的一个方案，它在选择性培养基中选择Trp+转化子。Yeast vectors often contain an origin of replication sequence, such as that from a 2μ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence, and a selectable marker gene. Suitable promoter sequences for yeast vectors include promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J Biol Chem 255:2073 (1980)) or other glycolytic enzymes (Holland et al. , Biochem 17:4900 (1978)) such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphate Glycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase, among others. Other suitable vectors and promoters for expression in yeast are further described in Fleer et al., Gene 107:285 (1991). Suitable promoters and vectors for other yeast and yeast transformation protocols are well known in the art. Yeast transformation protocols are also well known. Hinnen et al., Proc Natl Acad Sci 75:1929 (1978) describe such a protocol, which selects for Trp+ transformants in selective media.
任何真核细胞培养物都是可行的，无论是源自脊椎动物还是无脊椎动物的培养物。无脊椎动物细胞的例子，包括植物和昆虫细胞(Luckow等人,Bio/Technology 6:47(1988)；Miller等人,Genetic Engineering,Setlow等人,eds.,vol.8,pp.277-9,PlenumPublishing(1986)；以及Maeda等人,Nature 315:592(1985))。例如，杆状病毒系统可用于生产异源蛋白。在昆虫系统中，苜蓿银蚊夜蛾核型多角体病毒(Autographa californicanuclear polyhedrosis virus，AcNPV)可用作为载体用来表达外来基因。该病毒在草地贪夜蛾(Spodoptera frugiperda)细胞中生长。抗体编码序列可在AcNPV启动子(例如多角体蛋白启动子)的控制下被克隆。其它已经鉴定的宿主包括伊蚊(Aedes)、黑腹果蝇(Drosophila melanogaster)和家蚕(Bombyx mori)。多种各样的转染病毒株是公众可获得的，例如AcNPV的L-1变体和家蚕NPV的Bm-5株。此外，如本技术领域内众所周知，棉花、玉米、马铃薯、大豆、牵牛花(petunia)、西红柿和烟草的植物细胞培养物也可用作为宿主。Any eukaryotic cell culture is workable, whether of vertebrate or invertebrate origin. Examples of invertebrate cells include plant and insect cells (Luckow et al., Bio/Technology 6:47 (1988); Miller et al., Genetic Engineering, Setlow et al., eds., vol.8, pp.277-9 , Plenum Publishing (1986); and Maeda et al., Nature 315:592 (1985)). For example, the baculovirus system can be used to produce heterologous proteins. In insect systems, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be cloned under the control of an AcNPV promoter (eg, the polyhedrin promoter). Other identified hosts include Aedes, Drosophila melanogaster and Bombyx mori. A variety of transfection virus strains are publicly available, such as the L-1 variant of AcNPV and the Bm-5 strain of Bombyx mori NPV. In addition, plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco may also be used as hosts, as is well known in the art.
脊椎动物细胞、脊椎动物细胞在培养基(组织培养基)中的繁殖，可以是一种常规步骤，虽然苛求细胞系确实存在，它需要例如具有独特因子的专门培养基、滋养细胞等，参阅Tissue Culture,Kruse等人,eds.,Academic Press(1973)。有用的哺乳动物宿主细胞系的例子为猴肾；人胚肾系；幼仓鼠肾细胞；中国仓鼠卵巢细胞/-DHFR(CHO,Urlaub等人,ProcNatl Acad Sci USA 77:4216(1980))；小鼠塞尔托利细胞；人宫颈癌细胞(例如HeLa)；犬肾细胞；人肺细胞；人肝细胞；小鼠乳腺肿瘤；以及NS0细胞。Vertebrate cells, propagation of vertebrate cells in culture medium (tissue culture medium), can be a routine procedure, although fastidious cell lines do exist, which require e.g. specialized media with unique factors, feeder cells, etc., see Tissue Culture, Kruse et al., eds., Academic Press (1973). Examples of useful mammalian host cell lines are monkey kidney; human embryonic kidney line; baby hamster kidney cells; Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., ProcNatl Acad Sci USA 77:4216 (1980)); Murine Sertoli cells; human cervical cancer cells (eg HeLa); canine kidney cells; human lung cells; human hepatocytes; mouse breast tumors; and NSO cells.
宿主细胞用载体转化以产生抗体，并在常规营养培养基中培养，所述培养基含有生长因子、维生素、矿物质等，以及适合于所用细胞和载体的诱导物。常用的启动子序列和增强子序列是源自多瘤病毒、腺病毒2、猿猴病毒40(SV40)以及人的巨细胞病毒(CMV)。源自SV40病毒基因组的DNA序列可用于提供其它遗传元件(如SV40起点、早期和晚期启动子、增强子、剪接和多聚腺苷化位点)，以在哺乳动物宿主细胞中表达结构基因序列。病毒早期和晚期启动子是特别有用的，因为两者都很容易从病毒基因组作为片段获得，所述片段也可能含有病毒复制起点。用于哺乳动物宿主细胞的示例性表达载体可从商业渠道获得。Host cells are transformed with the vector to produce the antibody and cultured in a conventional nutrient medium containing growth factors, vitamins, minerals, etc., and inducers appropriate to the cells and vector used. Commonly used promoter sequences and enhancer sequences are those derived from polyoma virus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus (CMV). DNA sequences derived from the SV40 viral genome can be used to provide additional genetic elements (such as SV40 origin, early and late promoters, enhancers, splicing and polyadenylation sites) for expression of structural gene sequences in mammalian host cells . The viral early and late promoters are particularly useful because both are readily obtained from the viral genome as fragments that may also contain a viral origin of replication. Exemplary expression vectors for mammalian host cells are commercially available.
可从商业渠道获得的培养基如Ham's F10、极限必需培养基(MEM)、RPMI-1640和Dulbecco改良的Eagle培养基(DMEM)都适合于培养宿主细胞。此外，Ham等人,Meth Enzymol58:44(1979)和Barnes等人,Anal Biochem 102:255(1980)，以及US专利号4,767,704；4,657,866；4,560,655；5,122,469；5,712,163；或6,048,728中所述的任何培养基也可作为宿主细胞的培养基使用。这些培养基中任何一种，必要时都可补充激素和/或其它生长因子(如胰岛素，转铁蛋白或表皮生长因子)，盐(如氯化物，如氯化钠、氯化钙或氯化镁；以及磷酸盐)、缓冲液(如HEPES)、核苷酸(如腺苷和胸苷)、抗生素，痕量元素(定义为无机化合物，通常其存在的最终浓度在微摩尔的范围内)，以及葡萄糖或相当的能量来源。任何其它必要的补充，都可以适当的浓度包括在内，作为一项设计选择。培养条件，如温度、pH值等，都是本技术领域内已知适合于细胞的，并使转基因能够理想表达。Commercially available media such as Ham's F10, Minimal Essential Medium (MEM), RPMI-1640, and Dulbecco's Modified Eagle's Medium (DMEM) are suitable for culturing host cells. In addition, Ham et al., Meth Enzymol 58:44 (1979) and Barnes et al., Anal Biochem 102:255 (1980), and any of the media described in US Pat. Nos. 4,767,704; 4,657,866; 4,560,655; It can also be used as a culture medium for host cells. Any of these media, if necessary, may be supplemented with hormones and/or other growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as chlorides, such as sodium chloride, calcium chloride or magnesium chloride; and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics, trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and Glucose or equivalent energy source. Any other necessary supplements can be included at appropriate concentrations as a design choice. Culture conditions, such as temperature, pH, etc., are known in the art to be suitable for the cells and to allow the desired expression of the transgene.
通过本技术领域内任何已知的方法，就可获得目的多核苷酸，并确定该多核苷酸的核苷酸序列。例如，如果抗体的核苷酸序列是已知的，就可从化学合成的寡核苷酸装配编码抗体的多核苷酸(例如Kutmeier等人,Bio/Techniques 17:242(1994)所叙述的)，然后扩增连接的寡核苷酸，例如通过PCR。The polynucleotide of interest can be obtained by any known method in the art, and the nucleotide sequence of the polynucleotide can be determined. For example, if the nucleotide sequence of the antibody is known, polynucleotides encoding the antibody can be assembled from chemically synthesized oligonucleotides (such as described by Kutmeier et al., Bio/Techniques 17:242 (1994)) , and then amplify the ligated oligonucleotides, for example by PCR.
可选地，编码抗体的多核苷酸可从表达该抗体的细胞的核酸产生。如果含有编码某特定抗体的核酸的克隆是不可得的，但抗体分子的序列是已知的，则可从适当来源如文库获得编码免疫球蛋白的核酸，所述核酸也许是对产生抗体的细胞具有特异性的核酸，所述细胞如选择来表达本发明之抗体的杂交瘤细胞。可为PCR扩增配置适宜的引物。然后，使用本技术领域内众所周知的任何方法，可将PCR所产生的扩增核酸克隆到可复制的克隆载体中去。Alternatively, a polynucleotide encoding an antibody can be produced from nucleic acid of a cell expressing the antibody. If a clone containing nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, immunoglobulin-encoding nucleic acid may be obtained from an appropriate source, such as a library, which may be specific to the antibody-producing cell A nucleic acid having specificity, such as a hybridoma cell selected to express an antibody of the invention. Appropriate primers can be configured for PCR amplification. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.
一旦核苷酸序列和对应的抗体氨基酸序列被确定，就可使用本技术领域内已知的操纵核苷酸序列的方法，如DNA重组技术、定点诱变，PCR等(参阅，例如Sambrook等人,Molecular Cloning,A Laboratory Manual,2版，Cold Spring Harbor Laboratory(1990)；以及Ausubel等人,eds.,Current Protocols in Molecular Biology,JohnWiley&Sons(1998)，操纵该抗体的核苷酸序列以获得本文所述的目的等效物，产生含有不同氨基酸序列的抗体，例如，引起氨基酸取代、缺失和/或插入。Once the nucleotide sequence and corresponding antibody amino acid sequence have been determined, methods known in the art for manipulating the nucleotide sequence can be used, such as recombinant DNA techniques, site-directed mutagenesis, PCR, etc. (see, e.g., Sambrook et al. , Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory (1990); and Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1998), manipulated the nucleotide sequence of the antibody to obtain the nucleotide sequence described herein To obtain equivalents of interest as described above, antibodies are produced that contain different amino acid sequences, for example, resulting in amino acid substitutions, deletions and/or insertions.
可以众所周知的方法检查重链和/或轻链可变结构域的氨基酸序列，以鉴定CDR序列，例如，通过与其它重链和轻链可变区域的已知氨基酸序列的比较以确定序列高可变性的区域。使用常规DNA重组技术，可将一个或多个CDR插入构架区，例如插入人的构架区使非人源抗体人源化，如上所述。通过该构架区与一个或多个CDR的组合而产生的目的多核苷酸，可编码与IL-4和/或IL-13特异性结合的抗体或至少编码其ED结构域。例如，这种方法可用于产生一个或多个参与链内二硫键的可变区域半胱氨酸残基的氨基酸的取代或缺失，以产生缺乏一个或多个链内二硫键的抗体分子。The amino acid sequences of the heavy and/or light chain variable domains can be examined by well-known methods to identify the CDR sequences, for example, by comparison with known amino acid sequences of other heavy and light chain variable domains to determine sequence high likelihood. denatured area. Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region, eg, into a human framework region, to humanize a non-human antibody, as described above. The polynucleotide of interest produced by combining the framework region with one or more CDRs may encode an antibody specifically binding to IL-4 and/or IL-13 or at least encode its ED domain. For example, this method can be used to generate amino acid substitutions or deletions of one or more variable region cysteine residues that participate in intrachain disulfide bonds to generate antibody molecules that lack one or more intrachain disulfide bonds .
本发明的抗体或抗体片段可用来在体外或体内检测生物样品中的IL-4和/或IL-13，因此也就可以检测表达IL-4和/或IL-13的细胞。在一个实施方案中，本发明的抗IL-4和/或IL-13抗体被用来确定组织中或来自该组织的细胞中IL-4和/或IL-13的存在及水平。例如，在用本发明之抗体或抗体片段的免疫测定中，可测定IL-4和/或IL-13在该组织或活检样品中的水平。组织或其活检样品可冷冻或固定。同一方法或其它方法可用于测定IL-4和/或IL-13的其它性质，如它的水平、细胞定位、mRNA水平、其突变等等。Antibodies or antibody fragments of the present invention can be used to detect IL-4 and/or IL-13 in biological samples in vitro or in vivo, and thus cells expressing IL-4 and/or IL-13 can also be detected. In one embodiment, anti-IL-4 and/or IL-13 antibodies of the invention are used to determine the presence and level of IL-4 and/or IL-13 in a tissue or in cells from the tissue. For example, in an immunoassay using an antibody or antibody fragment of the invention, the level of IL-4 and/or IL-13 in the tissue or biopsy sample can be determined. Tissue or biopsy samples thereof may be frozen or fixed. The same method or other methods can be used to determine other properties of IL-4 and/or IL-13, such as its levels, cellular localization, mRNA levels, mutations thereof, and the like.
上述方法可用于例如在已知将要或疑患癌症的受试者中诊断癌症，其中将该患者中测量的IL-4和/或IL-13水平与正常参照受试者或标准相比。本发明的测定也可用于诊断关节炎或其它以B细胞的浸润和富集以及分化淋巴样组织发展为特征的自身免疫性疾病。The methods described above are useful, for example, in diagnosing cancer in a subject known to be or suspected of developing cancer, wherein IL-4 and/or IL-13 levels measured in the patient are compared to normal reference subjects or standards. The assays of the invention may also be used to diagnose arthritis or other autoimmune diseases characterized by infiltration and enrichment of B cells and the development of differentiated lymphoid tissue.
本发明还提供了为了研究或诊断应用而进一步标记的单克隆抗体、人源化抗体及其表位结合片段。在某些实施方案中，该标记是放射性标记的、荧光团、生色团、显像剂，或金属离子。The invention also provides monoclonal antibodies, humanized antibodies, and epitope-binding fragments thereof that are further labeled for research or diagnostic applications. In certain embodiments, the label is a radiolabel, a fluorophore, a chromophore, an imaging agent, or a metal ion.
还提供了一种诊断方法，其中将所述标记抗体或其表位结合片段施用给疑患癌症、关节炎、自身免疫性疾病或其它IL-4和/或IL-13介导的疾病的受试者，并且将所述标记在受试者体内的分布予以测量或监视。Also provided is a diagnostic method wherein the labeled antibody or epitope binding fragment thereof is administered to a subject suspected of having cancer, arthritis, autoimmune disease or other IL-4 and/or IL-13 mediated disease a subject, and the distribution of the marker in the subject is measured or monitored.
本发明之抗体及其片段可作为亲和纯化剂使用。在此过程中，使用本技术领域已知的方法，将抗体固定在固相上，如葡聚糖或琼脂糖树脂或滤纸上。被固定的抗体与含有IL-4和/或IL-13的样品或待纯化的载有所述IL-4和/或IL-13的细胞接触，然后用适宜的溶剂洗涤支持物，该溶剂将除去样品中除IL-4和/或IL-13或待纯化细胞以外几乎所有的物质，IL-4和/或IL-13或待纯化细胞则结合在被固定的本发明之抗体上。最后，用另一种适宜的溶剂(如甘氨酸缓冲液，pH 5.0)洗涤支持物，这将从抗体上释放IL-4和/或IL-13或细胞。Antibodies and fragments thereof of the present invention can be used as affinity purification agents. During this process, the antibodies are immobilized on a solid phase, such as dextran or agarose resin, or on filter paper, using methods known in the art. The immobilized antibody is contacted with the sample containing IL-4 and/or IL-13 or the cells carrying the IL-4 and/or IL-13 to be purified, and then the support is washed with a suitable solvent, which will Almost all substances in the sample except IL-4 and/or IL-13 or the cells to be purified are removed, and IL-4 and/or IL-13 or the cells to be purified are bound to the immobilized antibody of the present invention. Finally, the support is washed with another suitable solvent (eg, glycine buffer, pH 5.0), which will release IL-4 and/or IL-13 or cells from the antibody.
为了诊断应用，目的抗体通常会用可检测的部分加以标记。有许多标记可供利用，通常可分为以下几类：(a)放射性同位素，如36S、14C、125I、3H和131I(抗体可用放射性同位素标记，例如使用Current Protocols in Immunology,vol.12,Coligen等人,编辑，Wiley-Interscience,New York(1991)所述的技术，并且放射性可用闪烁计数来测量)；(b)荧光标记，如稀土螯合物(铕螯合物)、荧光素及其衍生物、罗丹明及其衍生物、丹磺酰、丽丝胺、藻红蛋白以及得克萨斯红；荧光标记可用例如CurrentProtocols in Immunology(同上)披露的技术缀合到抗体上，其中荧光可用荧光计定量；以及(c)有多种酶底物标记可供利用(US专利号4,275,149提供了综述)，酶通常可催化生色底物的化学改变，这可采用多种技术来测量，例如酶可催化底物颜色的变化，这可用分光光度来衡量；或者，酶可以改变底物的荧光或化学发光。定量荧光变化的技术是众所周知的，例如使用发光计，或者该标记可向荧光接受体提供能量。酶标记的例子包括荧光素酶(例如，萤火虫荧光素酶和细菌荧光素酶；US专利号4,737,456)、萤光素(luciferin)、2,3-dihydrophthalazinedione、苹果酸脱氢酶、尿素酶，过氧化物酶如辣根过氧化物酶(HRPO)、碱性磷酸酶，β-半乳糖苷酶、葡糖淀粉酶、溶菌酶、糖氧化酶(如葡糖氧化酶、半乳糖氧化酶和葡糖-6-磷酸脱氢酶)、杂环氧化酶(如尿酸酶和黄嘌呤氧化酶)、乳过氧化物酶(lactoperoxiase)、微过氧化物酶(microperoxiase)等。使酶与抗体缀合的技术在O'Sullivan等人,Meth Enz,ed.Langone&Van Vunakis,Academic Press,New York,73(1981)中有所叙述。For diagnostic applications, the antibody of interest will usually be labeled with a detectable moiety. A number of labels are available and generally fall into the following categories: (a) Radioactive isotopes such as36 S,14 C,125 I,3 H, and131 I (antibodies can be labeled with radioactive isotopes, for example using Current Protocols in Immunology, vol.12, Coligen et al., eds. Wiley-Interscience, New York (1991) described technique, and radioactivity can be measured by scintillation counting); (b) fluorescent labels, such as rare earth chelates (europium chelates) , fluorescein and its derivatives, rhodamine and its derivatives, dansyl, lissamine, phycoerythrin, and Texas Red; fluorescent labels can be conjugated to antibodies using techniques such as those disclosed in Current Protocols in Immunology (supra), wherein Fluorescence can be quantified with a fluorometer; and (c) a variety of enzyme substrate labels are available (reviewed in US Pat. No. 4,275,149), and enzymes often catalyze chemical changes in chromogenic substrates, which can be measured using a variety of techniques For example, an enzyme can catalyze a change in the color of a substrate, which can be measured spectrophotometrically; alternatively, an enzyme can change the fluorescence or chemiluminescence of a substrate. Techniques for quantifying changes in fluorescence are well known, such as using a luminometer, or the label can provide energy to a fluorescent acceptor. Examples of enzyme labels include luciferase (e.g., firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinedione, malate dehydrogenase, urease, Oxidases such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase and glucose Sugar-6-phosphate dehydrogenase), heterocyclic oxidase (such as uricase and xanthine oxidase), lactoperoxidase (lactoperoxidase), microperoxidase (microperoxidase) and so on. Techniques for conjugating enzymes to antibodies are described in O'Sullivan et al., Meth Enz, ed. Langone & Van Vunakis, Academic Press, New York, 73 (1981).
当使用这些标记时，有适宜的底物可供利用，例如：(i)对于辣根过氧化物酶可用氢过氧化物酶(hydrogen peroxiase)作为底物，其中氢过氧化物酶氧化染料前体(例如邻苯二胺(OPD)或3,3',5,5'-四甲基联苯胺氢氯化物(TMB))；(ii)对于碱性磷酸酶(AP)可用对硝基苯基磷酸酯作为生色底物；以及(iii)β-D-半乳糖苷酶(β-D-Gal)可用生色底物(例如对硝基苯基-β-D-半乳糖苷酶)，或荧光底物如4-甲基伞形基酮酰-β-D-半乳糖苷酶。When using these labels, suitable substrates are available, for example: (i) Hydrogen peroxidase (hydrogen peroxidase) can be used as a substrate for horseradish peroxidase, wherein hydrogen peroxidase oxidizes the dye precursor (e.g. o-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine hydrochloride (TMB)); (ii) p-nitrophenyl for alkaline phosphatase (AP) base phosphate as a chromogenic substrate; and (iii) β-D-galactosidase (β-D-Gal) can use a chromogenic substrate (e.g. p-nitrophenyl-β-D-galactosidase) , or fluorescent substrates such as 4-methylumbelliferylketoacyl-β-D-galactosidase.
其它酶-底物组合可供本技术领域专业人员利用。对于一般性评述，可参阅US专利号4,275,149和4,318,980。Other enzyme-substrate combinations are available to those skilled in the art. For general comments, see US Patent Nos. 4,275,149 and 4,318,980.
有时，标记与抗体是间接地缀合。例如，抗体可与生物素缀合，上述任何报导子都可与抗生物素蛋白缀合，反之亦然。生物素与抗生物素蛋白选择性地结合，因此，标记可与抗体以该间接方式缀合。或者，为了实现标记的间接缀合，抗体与小的半抗原(如地高辛)缀合，上述不同类型的标记或报导子之一与抗地高辛抗体缀合。因此，使用第二种抗体可以实现标记与抗体或突变蛋白的间接缀合。Sometimes the label is conjugated to the antibody indirectly. For example, the antibody can be conjugated to biotin, and any of the reporters described above can be conjugated to avidin, and vice versa. Biotin binds selectively to avidin, so a label can be conjugated to the antibody in this indirect manner. Alternatively, to achieve indirect conjugation of a label, the antibody is conjugated to a small hapten such as digoxin, and one of the different types of labels or reporters described above is conjugated to an anti-digoxigenin antibody. Thus, indirect conjugation of the label to the antibody or mutein can be achieved using a second antibody.
在本发明的另一个实施方案中，抗体不必加以标记，它的存在可使用与抗体(另一种形式的第二抗体)结合的标记抗体来检测。In another embodiment of the invention, the antibody need not be labeled and its presence can be detected using a labeled antibody bound to the antibody (another form of secondary antibody).
本发明之抗体可用于任何已知的测定方法，如竞争性结合测定、直接和间接夹心测定以及免疫沉淀测定。Zola,Monoclonal Antibodies：A Manual of Techniques(CRCPress,Inc.1987)。The antibodies of the invention can be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of Techniques (CRC Press, Inc. 1987).
竞争性结合测定取决于带标记的标准物与试验样品在与有限量抗体结合中的竞争能力。试验样品中抗原的量与跟抗体结合的标准物的量成反比。为便于测定结合的标准物的量，抗体在竞争之前或之后通常是不溶的。结果，与抗体结合的标准物和试验样品可与未结合的标准物和试验样品方便地分离。Competitive binding assays depend on the ability of a labeled standard to compete with a test sample for binding to a limited amount of antibody. The amount of antigen in the test sample is inversely proportional to the amount of standard bound to the antibody. To facilitate determination of the amount of standard bound, antibodies are usually insoluble either before or after competition. As a result, antibody-bound standards and test samples can be conveniently separated from unbound standards and test samples.
夹心测定涉及使用两种抗体，每一种都有能力与待检测靶的不同免疫原部分(决定子或表位)结合。在夹心测定中，待分析试验样品与直接或间接固定在固体支持物上的第一种抗体结合，然后直接或间接地加以标记的第二种抗体与已经结合的试验样品结合，从而形成不溶性三部分复合物，参阅例如US专利号4,376,110。第二种抗体本身可用可检测的部分加以标记(直接夹心测定)，或可用抗免疫球蛋白抗体或用可检测部分加以标记的结合对(例如抗体/抗原、受体/配体、酶/底物)的其它适宜成员测量(间接夹心测定)。例如，一种类型的夹心测定是ELISA测定，在这种情况下，该可检测部分是酶。Sandwich assays involve the use of two antibodies, each capable of binding to a different immunogenic portion (determinant or epitope) of the target to be detected. In a sandwich assay, the test sample to be analyzed is bound to a first antibody, which is directly or indirectly immobilized on a solid support, and a second labeled antibody, directly or indirectly, is then bound to the already bound test sample to form an insoluble three Part of the complex, see eg US Patent No. 4,376,110. The second antibody can itself be labeled with a detectable moiety (direct sandwich assay), or it can be an anti-immunoglobulin antibody or a binding pair labeled with a detectable moiety (e.g. antibody/antigen, receptor/ligand, enzyme/substrate). other suitable member measurement (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.
本发明还包括试剂试剂盒，例如其包括抗体、其片段、同源物、其衍生物等，例如带标记或具有细胞毒性的辍合物，以及抗体使用说明书、杀死特定类型细胞的辍合物等等。该说明书可包括在体外、体内或离体使用抗体、辍合物等的指导。抗体可以是液体形式或固体，通常是冻干的。该试剂盒可包含其它适宜的试剂，如缓冲液、重构溶液以及为了预定用途的其它必要成分。考虑了以预定量包装好的试剂组合与用于其用途的说明书，所述用途例如用于治疗用途或用于进行诊断测定。当抗体是带标记的时，例如用酶标记的，那么该试剂盒可包括底物和酶所需的辅因子(例如提供可检测生色团或荧光团的底物前体)。此外，其它添加剂，如稳定剂、缓冲液(例如封闭缓冲液或裂解缓冲液)等也可包括在内。多种试剂的相对量可以改变而提供试剂溶液的浓缩物，这就提供了用户灵活性、节省空间、节省试剂等。这些试剂也可以干粉形式提供，通常是冻干形式，包括赋形剂，它在溶解时可提供具有适当浓度的试剂溶液。The invention also includes kits of reagents, for example comprising antibodies, fragments thereof, homologues, derivatives thereof, etc., such as labeled or cytotoxic conjugates, as well as instructions for use of antibodies, conjugates that kill specific types of cells, things and so on. The instructions may include directions for using the antibodies, conjugates, etc. in vitro, in vivo, or ex vivo. Antibodies can be in liquid form or solid, usually lyophilized. The kit may contain other suitable reagents such as buffers, reconstitution solutions and other components necessary for the intended use. Reagents packaged in predetermined quantities are contemplated in combination with instructions for their use, eg, for therapeutic use or for performing a diagnostic assay. When the antibody is labeled, eg, with an enzyme, the kit may include a substrate and cofactors required for the enzyme (eg, a substrate precursor providing a detectable chromophore or fluorophore). In addition, other additives such as stabilizers, buffers (eg, blocking buffer or lysis buffer), etc. may also be included. The relative amounts of various reagents can be varied to provide a concentration of reagent solutions, which provides user flexibility, space savings, reagent savings, and the like. These reagents may also be provided in dry powder form, usually lyophilized form, including excipients which, on dissolution, will provide a solution of the reagents at the appropriate concentration.
本发明之抗体可用于治疗哺乳动物。在一个实施方案中，例如，出于获得临床前数据的目的，将目的抗体或等效物施用给非人哺乳动物。示例性的待治疗非人哺乳动物，包括非人灵长类动物、狗、猫、啮齿类动物以及其它哺乳动物，其中进行了临床前研究。这种哺乳动物可为需用抗体治疗的疾病的建立的动物模型，或可用于研究目的抗体的毒性。在每个这样的实施方案中，可在哺乳动物中进行剂量递增研究。Antibodies of the invention can be used to treat mammals. In one embodiment, the antibody of interest or equivalent is administered to a non-human mammal, eg, for the purpose of obtaining preclinical data. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents, and other mammals in which preclinical studies have been conducted. Such a mammal can be an established animal model of a disease requiring treatment with the antibody, or can be used to study the toxicity of the antibody of interest. In each of these embodiments, dose escalation studies can be performed in mammals.
抗体，无论有无第二个组分(如与之缀合的治疗剂部分)，无论是单独或与细胞毒性因子组合施用，均可作为治疗剂使用。本发明涉及以抗体为基础的疗法，其中保留给动物、哺乳动物或人施用本发明之抗体，以治疗IL-4和/或IL-13介导的疾病、障碍或状况。Antibodies, with or without a second component (eg, a therapeutic moiety to which they are conjugated), whether administered alone or in combination with a cytotoxic factor, can be used as therapeutic agents. The present invention relates to antibody-based therapies wherein administration of antibodies of the present invention to animals, mammals or humans is reserved for the treatment of IL-4 and/or IL-13 mediated diseases, disorders or conditions.
本发明所使用的术语"治疗"是指治疗性治疗和预防性或预防措施。它指的是预防、治愈、逆转、减弱、改善、最小化、抑制或停止疾病状态、疾病进程、疾病致病因素(如细菌或病毒)或其它异常状况的有害效应。The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventive measures. It refers to preventing, curing, reversing, attenuating, ameliorating, minimizing, inhibiting or stopping the deleterious effects of a disease state, disease process, disease causative agent (such as bacteria or virus) or other abnormal condition.
因此，本发明还包含多价抗体，包括双特异抗IL-4/IL-13抗体，其具有与之相连的诊断或治疗功能的效应分子、原子或其它物质。例如，抗体可具有放射性诊断标签或放射性细胞毒性原子或金属或细胞毒性物质如蓖麻毒蛋白链，与之相连用于癌症的体内诊断或治疗。Accordingly, the invention also encompasses multivalent antibodies, including bispecific anti-IL-4/IL-13 antibodies, having associated therewith effector molecules, atoms or other substances for diagnostic or therapeutic function. For example, antibodies may have radioactive diagnostic labels or radioactive cytotoxic atoms or metals or cytotoxic substances such as ricin chains attached thereto for in vivo diagnosis or treatment of cancer.
此外，本发明的抗体还可用于免疫测定、纯化方法以及其它用到免疫球蛋白或其片段的方法。此类用途在本领域为人所熟知。In addition, the antibodies of the invention can be used in immunoassays, purification methods, and other methods using immunoglobulins or fragments thereof. Such uses are well known in the art.
相应地，本发明还提供包含本发明的抗IL-13和/或抗IL-4的抗体或其片段的组合物，所述抗体方便地和可药用的载体、稀释剂或赋形剂组合，这是本领域的常规做法。Accordingly, the present invention also provides compositions comprising the anti-IL-13 and/or anti-IL-4 antibodies or fragments thereof of the present invention, which are conveniently combined with pharmaceutically acceptable carriers, diluents or excipients , which is a common practice in this field.
本发明所使用的术语"药物组合物"系指多种制备物的制剂。含有治疗有效量的多价抗体的制剂为无菌液体溶液、液体悬浮剂或冻干形式，任选地包含稳定剂或赋形剂。The term "pharmaceutical composition" as used herein refers to a preparation of various preparations. Formulations containing therapeutically effective amounts of multivalent antibodies are in sterile liquid solution, liquid suspension or lyophilized form, optionally containing stabilizers or excipients.
本发明所使用的术语"障碍"系指任何能够得益于本发明抗体治疗的状况。这包括慢性和急性障碍或疾病，包括那些倾向于使哺乳动物，尤其是人染上所述障碍的病理状况。本文中非限制性的有待治疗的障碍之实例包括癌症、炎症、自身免疫疾病、感染、心血管疾病、呼吸疾病、神经疾病以及代谢性疾病。The term "disorder" as used herein refers to any condition that would benefit from treatment with the antibodies of the invention. This includes chronic and acute disorders or diseases, including those pathological conditions which tend to predispose mammals, especially humans, to said disorders. Non-limiting examples of disorders to be treated herein include cancer, inflammation, autoimmune disease, infection, cardiovascular disease, respiratory disease, neurological disease, and metabolic disease.
本发明的抗体可用来治疗、抑制或预防疾病，如变应性疾病、Th2介导的疾病、IL-13介导的疾病、IL-4介导的疾病和/或IL-4/IL-13介导的疾病。此类疾病的实例包括霍奇金病、哮喘、变应性哮喘、特应性皮炎、特应性变态反应、溃疡性结肠炎、硬皮病、变应性鼻炎、COPD3特发性肺纤维化、慢性移植排斥、博来霉素诱导的肺纤维化、辐射诱导的肺纤维化、肺部肉芽肿、进行性系统性硬化、血吸虫病、肝纤维化、肾癌、伯基特淋巴瘤、霍奇金病、非-霍奇金病、塞扎里综合征、哮喘、浓毒性关节炎、疱疹样皮炎、慢性特发性荨麻疹、溃疡性结肠炎、硬皮病、肥厚性疤痕、Whipple病、良性前列腺增生、IL-4受体起作用的肺部障碍、IL-4受体介导的上皮屏障破坏起作用的状况、IL-4受体起作用的消化系统障碍、对药物的变应性反应、川崎病、镰状细胞病、Churg-Strauss综合征、Grave's病、先兆子痫、Sjogren's综合征、自身免疫性淋巴组织增生综合征、自身免疫性溶血性贫血、Barrett's食管、自身免疫性葡萄膜炎、肺结核、囊性纤维化、变应性支气管肺真菌病、慢性阻塞性肺病、博来霉素诱导的肺病和纤维化、肺泡蛋白沉积症(pulmonary alveolar proteinosis)、成人呼吸窘迫综合征、结节病、高IgE综合征、特发性嗜伊红细胞增多综合征(idiopathic hypereosinophilsyndrome)、自身免疫性发疱病(autoimmune blistering disease)、寻常型天疱疮、大疱性类天疱疮、重症肌无力、慢性疲劳综合征、肾病。The antibodies of the present invention can be used to treat, inhibit or prevent diseases, such as allergic diseases, Th2-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases and/or IL-4/IL-13 mediated disease. Examples of such diseases include Hodgkin's disease, asthma, allergic asthma, atopic dermatitis, atopic allergy, ulcerative colitis, scleroderma, allergic rhinitis, COPD3 idiopathic pulmonary fibrosis , chronic transplant rejection, bleomycin-induced pulmonary fibrosis, radiation-induced pulmonary fibrosis, pulmonary granuloma, progressive systemic sclerosis, schistosomiasis, liver fibrosis, renal carcinoma, Burkitt lymphoma, cholangiocarcinoma Chiggin's disease, non-Hodgkin's disease, Sezary syndrome, asthma, erythrotoxic arthritis, dermatitis herpetiformis, chronic idiopathic urticaria, ulcerative colitis, scleroderma, hypertrophic scarring, Whipple's disease , benign prostatic hyperplasia, pulmonary disorders in which IL-4 receptors play a role, conditions in which IL-4 receptor-mediated epithelial barrier disruption plays a role, digestive disorders in which IL-4 receptors play a role, reactions to drugs Sexual reaction, Kawasaki disease, sickle cell disease, Churg-Strauss syndrome, Grave's disease, preeclampsia, Sjogren's syndrome, autoimmune lymphoproliferative syndrome, autoimmune hemolytic anemia, Barrett's esophagus, autoimmune Uveitis, tuberculosis, cystic fibrosis, allergic bronchopulmonary mycosis, chronic obstructive pulmonary disease, bleomycin-induced lung disease and fibrosis, pulmonary alveolar proteinosis, adult respiratory distress syndrome , sarcoidosis, hyper-IgE syndrome, idiopathic hypereosinophil syndrome, autoimmune blistering disease, pemphigus vulgaris, bullous pemphigoid, Myasthenia gravis, chronic fatigue syndrome, kidney disease.
术语"变应性疾病"系指病理状况，其中患者对一种正常情况下非免疫原性的物质超敏并作出免疫反应。变应性疾病的一般特征是IgE激活肥大细胞而造成炎症反应(如局部反应、全身反应)，这类反应可导致如流鼻涕那样的良性症状，也可能是危及生命的过敏性休克和死亡。变应性疾病的实例包括但不限于变应性鼻炎(如花粉症)、哮喘(如变应性哮喘)、变应性皮炎(如湿疹)、接触性皮炎、食物过敏和荨麻疹(荨麻疹(hive))。The term "allergic disease" refers to a pathological condition in which a patient is hypersensitized and immune-reacted to a normally non-immunogenic substance. Allergic disease is generally characterized by IgE activation of mast cells leading to an inflammatory response (eg, local, systemic) that can lead to benign symptoms such as runny nose or life-threatening anaphylactic shock and death. Examples of allergic diseases include, but are not limited to, allergic rhinitis (such as hay fever), asthma (such as allergic asthma), allergic dermatitis (such as eczema), contact dermatitis, food allergies, and urticaria (urticaria (hive)).
此处所使用的"Th2介导的疾病"系指一种疾病，其病理是(全部或部份)由CD4+Th2T淋巴样细胞调控的免疫反应(Th2型免疫反应)造成的，其特征地形成IL-4、IL-5、IL-9和IL-13。Th2型免疫反应与某些细胞因子(如IL-4,IL-13)和某些类别的抗体(如IgE)的产生以及体液免疫有关。Th2-介导的疾病的特征是Th2细胞因子(如IL-4,IL-13)和/或某些类别的抗体(如IgE)的升高水平的存在，包括例如变应性疾病(如变应性鼻炎、特发性皮炎、哮喘(如特发性哮喘)、变应性气道疾病(AAD)、过敏性休克、结膜炎)，与IL-4和/或IL-13水平增高有关的自身免疫障碍(如类风湿性关节炎、宿主vs移植物疾病、肾病(如肾病综合征、狼疮性肾炎))，以及与IL-4和/或IL-13水平升高有关的感染(如病毒、寄生虫、真菌(如白色念珠菌(C.albicans))感染)。有些癌症与IL-4和/或IL-13的升高水平有关，或与IL-4诱导的和/或IL-13诱导的癌细胞增殖有关(如B细胞淋巴瘤、T细胞淋巴瘤、多发性骨髓瘤、头颈癌、乳腺癌和卵巢癌)。这些癌症可用本发明的Ii gaud治疗、抑制或预防。As used herein, "Th2-mediated disease" refers to a disease whose pathology is caused (in whole or in part) by an immune response mediated by CD4+ Th2 T lymphoid cells (Th2-type immune response), characterized by the formation of IL-4, IL-5, IL-9 and IL-13. Th2-type immune responses are related to the production of certain cytokines (such as IL-4, IL-13) and certain classes of antibodies (such as IgE) and humoral immunity. Th2-mediated diseases are characterized by the presence of elevated levels of Th2 cytokines (e.g. IL-4, IL-13) and/or certain classes of antibodies (e.g. IgE), including for example allergic diseases (e.g. allergic rhinitis, idiopathic dermatitis, asthma (such as idiopathic asthma), allergic airway disease (AAD), anaphylactic shock, conjunctivitis), associated with increased levels of IL-4 and/or IL-13 Autoimmune disorders (eg, rheumatoid arthritis, host vs graft disease, renal disease (eg, nephrotic syndrome, lupus nephritis)), and infections associated with elevated levels of IL-4 and/or IL-13 (eg, viral , parasites, fungi (such as Candida albicans (C.albicans)) infection). Some cancers are associated with elevated levels of IL-4 and/or IL-13, or IL-4-induced and/or IL-13-induced proliferation of cancer cells (eg, B-cell lymphoma, T-cell lymphoma, multiple myeloma, head and neck cancer, breast and ovarian cancer). These cancers can be treated, inhibited or prevented with the li gaud of the present invention.
本发明所使用的术语"癌症"系指或描述了哺乳动物尤其是人的生理状况，其典型特点是细胞无调控地生长。癌症的实例包括但不限于癌(carcinoma)、淋巴瘤、母细胞瘤、肉瘤和白血病。The term "cancer" as used herein refers to or describes the physiological condition in mammals, especially humans, which is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
本发明所使用的术语"自身免疫疾病"系指由于个体自身组织并针对自身组织的非恶性疾病或障碍。自身免疫疾病或障碍的实例包括但不限于炎症性反应如炎症性皮肤病，包括银屑病和皮炎；变应性状况如湿疹和哮喘；其它涉及T细胞浸润和慢性炎症反应的症状；动脉粥样硬化、糖尿病(如I型糖尿病或胰岛素依赖型糖尿病)；多发性硬化和中枢神经系统炎症性障碍。The term "autoimmune disease" as used herein refers to a non-malignant disease or disorder due to and against an individual's own tissues. Examples of autoimmune diseases or disorders include, but are not limited to, inflammatory responses such as inflammatory skin diseases, including psoriasis and dermatitis; allergic conditions such as eczema and asthma; other conditions involving T cell infiltration and chronic inflammatory responses; atherosclerosis diabetes mellitus (such as type 1 diabetes or insulin-dependent diabetes mellitus); multiple sclerosis and inflammatory disorders of the central nervous system.
本发明的抗体可以作为单独施用的组合物使用，或可与其它活性剂联合使用。抗体可以与现有的IL-13疗法(例如现有的IL-13活性剂如抗IL-13Rαl、IL-4/13Trap、抗IL-13)加抗IL-4抗体，以及现有的IL-4活性剂(如抗IL-4R、IL-4突变蛋白质、IL-4/13Trap)加抗IL-13抗体和IL-4抗体(如WO05/0076990(CAT),WO03/092610(Regeneron),WO00/64944(Genetic Inst.)以及WO2005/062967(Tanox))一起用于组合疗法中。Antibodies of the invention may be used as compositions administered alone, or may be used in combination with other active agents. Antibodies can be combined with existing IL-13 therapies (eg, existing IL-13 active agents such as anti-IL-13Rα1, IL-4/13Trap, anti-IL-13) plus anti-IL-4 antibodies, and existing IL-13 4 Active agents (such as anti-IL-4R, IL-4 mutein, IL-4/13Trap) plus anti-IL-13 antibody and IL-4 antibody (such as WO05/0076990 (CAT), WO03/092610 (Regeneron), WO00 /64944 (Genetic Inst.) and WO2005/062967 (Tanox)) are used together in combination therapy.
本发明的抗体可以与一个或多个另外的治疗或活性剂一起施用和/或配制。当一个配体与另外的治疗剂一起施用时，配体可在另外的治疗剂施用前、同时或施用后施用。一般来说，配体和另外的活性剂通常是提供治疗效果重叠的方式施用。能够与本发明的配体一起施用或一起配制的另外活性剂包括例如多种免疫治疗药物，诸如环孢素、甲氨蝶呤、阿霉素或顺铂、抗生素、抗真菌剂、抗病毒剂和免疫毒素。例如，当施用拮抗剂来预防、抑制或治疗肺部炎症或呼吸疾病时(如哮喘)，拮抗剂可与下述联合施用：磷酸二酯酶抑制剂(如磷酸二酯酶4抑制剂)、支气管扩张药(如β2激动剂、抗胆碱能药、茶碱)、短效β激动剂(如沙丁胺醇(albuterol)、舒喘灵(salbuiamol)、班布特罗、非诺特罗[sigmal]l、isoetherine、异丙肾上腺素、左旋沙丁胺醇(leva[iota]buterol)、澳西那林、吡布特罗、特布他林和tornlate)、长效β激动剂(如福莫特罗和沙美特罗)、短效抗胆碱能药(如异丙托溴铵和氧托溴铵)、长效抗胆碱能药(如噻托溴铵)、茶碱(如短效制剂、长效制剂)、吸入式类固醇(如倍氯米松、倍氯米松、布地奈德、氟尼缩松、丙酸氟替卡松和曲安西龙)、口服类固醇(如甲泼尼龙、泼尼松龙、prednisolon和泼尼松)、短效β激动剂与抗胆碱能药组合(如沙丁胺醇/舒喘灵/异丙托溴铵以及非诺特罗/异丙托溴铵)、长效贝塔激动剂与吸入式类固醇组合(如沙美特罗/氟替卡松以及福莫特罗/布地奈德)，以及溶粘蛋白剂(如厄多司坦、乙酰半胱氨酸、溴己新、carbocyslcine、guiiafencsin和碘化甘油。Antibodies of the invention may be administered and/or formulated with one or more additional therapeutic or active agents. When a ligand is administered with an additional therapeutic agent, the ligand can be administered before, simultaneously with, or after the administration of the additional therapeutic agent. In general, the ligand and the additional active agent are usually administered in a manner that provides an overlapping therapeutic effect. Additional active agents that can be administered or co-formulated with the ligands of the invention include, for example, various immunotherapeutic drugs such as cyclosporine, methotrexate, doxorubicin or cisplatin, antibiotics, antifungals, antivirals and immunotoxins. For example, when an antagonist is administered to prevent, suppress or treat lung inflammation or a respiratory disease (such as asthma), the antagonist can be administered in combination with a phosphodiesterase inhibitor (such as a phosphodiesterase 4 inhibitor), Bronchodilators (eg, beta2-agonists, anticholinergics, theophylline), short-acting beta-agonists (eg, albuterol, salbuiamol, bambuterol, fenoterol [sigmal] l, isoetherine, isoproterenol, leva[iota]buterol (leva[iota]buterol), ocenaline, pirbuterol, terbutaline and tornlate), long-acting beta agonists (such as formoterol and samemet) Terol), short-acting anticholinergics (such as ipratropium bromide and oxitropium bromide), long-acting anticholinergics (such as tiotropium bromide), theophylline (such as short-acting preparations, long-acting preparations), inhaled steroids (such as beclomethasone, beclomethasone, budesonide, flunisolide, fluticasone propionate, and triamcinolone), oral steroids (such as methylprednisolone, prednisolone, prednisolon, and prednisolone Nisone), combinations of short-acting beta-agonists and anticholinergics (such as albuterol/albuterol/ipratropium bromide and fenoterol/ipratropium bromide), long-acting beta-agonists and inhaled Steroid combinations (eg, salmeterol/fluticasone and formoterol/budesonide), and mucolytics (eg, erdosteine, acetylcysteine, bromhexine, carbocyslcine, guiiafencsin, and iodized glycerol.
其它能够与本发明的抗体共同施用以预防、抑制或治疗哮喘(如变应性哮喘)的合适的共治疗剂包括皮质类固醇(如倍氯米松、布地奈德、氟替卡松)、色苷酸盐、奈多罗米、β激动剂(如舒喘灵、特布他林、班布特罗、非诺特罗、茶丙特罗、tolubuterol、沙美特罗、fomtero)、扎鲁司特、沙美特罗、泼尼松、泼尼松龙、茶碱、zileutron、孟鲁司特以及白三烯改性剂。本发明的配体可以与很多适合治疗疾病(如Th-2介导的疾病、YL-A介导的疾病、IL-13介导的疾病、IL-4介导的疾病以及癌症)的共治疗剂共同施用，包括细胞因子、镇痛药/解热药、止吐剂以及化疗药物。Other suitable co-therapeutic agents that can be co-administered with the antibodies of the invention to prevent, suppress or treat asthma (such as allergic asthma) include corticosteroids (such as beclomethasone, budesonide, fluticasone), chromylates, Nedocromil, beta agonists (eg, albuterol, terbutaline, bambuterol, fenoterol, teaproterol, tolubuterol, salmeterol, fomterol), zafirlukast, salmeterol , prednisone, prednisolone, theophylline, zileutron, montelukast, and leukotriene modifiers. The ligand of the present invention can be used with many co-treatments suitable for treating diseases (such as Th-2-mediated diseases, YL-A-mediated diseases, IL-13-mediated diseases, IL-4-mediated diseases and cancer) Co-administration of various agents, including cytokines, analgesics/antipyretics, antiemetics, and chemotherapy drugs.
如本技术领域内众所周知或本文所述，本发明之抗体可以可药用的组合物形式提供。"生理上可接受的"、"药理学上可接受的"等术语意为已经联邦或州政府的监管机构批准，或已列入美国药典或其它普遍公认的用于动物尤其是人的药典。Antibodies of the invention may be provided in pharmaceutically acceptable compositions, as is well known in the art or as described herein. The terms "physiologically acceptable", "pharmacologically acceptable" and the like mean approved by a regulatory agency of the federal or state government, or listed in the US Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, especially humans.
可以任何可接受的方式对哺乳动物，尤其是人施用抗IL-4、抗IL-13以及双特异抗IL-4/IL-13抗体。输入方法包括但不限于胃肠外、皮下、腹膜内、肺内、鼻内、硬膜外、吸入和口服途径；若欲进行免疫抑制治疗，则病灶内(intralesional)施用。胃肠外输注包括肌内、皮内、静脉内、动脉内或腹膜内施用。抗体或组合物可以任何方便途径施用，例如通过输注或快速注射、通过上皮或粘膜衬里(例如口腔粘膜、直肠和肠道粘膜等)吸收，并可与其它生物活性剂一起施用。施用可以是全身性或局部性。此外，也许需要以任何适当途径将本发明的治疗抗体或组合物引入中枢神经系统，包括心室内和鞘内注射；心室内注射借助于一心室内导管，例如与一个贮器如奥莫耶贮器(Ommaya reservoir)连接的导管。此外，该抗体适合于以脉冲输注方式给药，尤其是以抗体剂量递减的方式。优选地，以注射方式给药，优选地静脉内注射或皮下注射，部分地取决于给药是短期还是长期。Anti-IL-4, anti-IL-13 and bispecific anti-IL-4/IL-13 antibodies can be administered to mammals, especially humans, in any acceptable manner. Methods of infusion include, but are not limited to, parenteral, subcutaneous, intraperitoneal, intrapulmonary, intranasal, epidural, inhalation, and oral routes; if immunosuppressive therapy is desired, intralesional administration. Parenteral infusions include intramuscular, intradermal, intravenous, intraarterial or intraperitoneal administration. Antibodies or compositions may be administered by any convenient route, such as by infusion or bolus injection, absorbed through epithelial or mucosal linings (eg, oral mucosa, rectal and intestinal mucosa, etc.), and may be administered with other biologically active agents. Administration can be systemic or localized. In addition, it may be desirable to introduce a therapeutic antibody or composition of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection is by means of an intraventricular catheter, for example, with a reservoir such as the Omoyer reservoir ( Ommaya reservoir) connected conduit. In addition, the antibodies are suitable for administration by pulse infusion, especially in tapered antibody doses. Preferably, the administration is by injection, preferably intravenous or subcutaneous injection, depending in part on whether the administration is short-term or chronic.
多种其它递送系统是众所周知的，可用于本发明之抗体的给药，包括例如封装于脂质体、微粒、微胶囊(参阅Langer,Science 249:1527(1990)；Treat等人,in Liposomesin the Therapy of Infectious Disease and Cancer；Lopez-Berestein等人,编辑,p.353-365(1989)；以及Lopez-Berestein,ibid.,p.317-327)以及能表达该化合物的重组细胞；受体介导的胞吞(参阅Wu等人,J Biol Chem 262:4429(1987))；核酸作为逆转录病毒或其它载体等的部分的构建。A variety of other delivery systems are well known and can be used to administer the antibodies of the invention, including, for example, encapsulation in liposomes, microparticles, microcapsules (see Langer, Science 249:1527 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer; Lopez-Berestein et al., ed., p.353-365 (1989); and Lopez-Berestein, ibid., p.317-327) and recombinant cells expressing the compound; receptor mediated Induced endocytosis (see Wu et al., J Biol Chem 262:4429 (1987)); construction of nucleic acids as part of retroviruses or other vectors and the like.
活性成分也可封装在例如以coascervation技术或界面多聚化制备的微胶囊中，例如分别在胶体药物递送系统(例如脂质体、白蛋白微球、微乳液、纳米粒子和纳米胶囊)或微乳液中的羟甲基纤维素或明胶微胶囊和聚-(甲基methacylate)微胶囊。Remington'sPharmaceutical Sciences(雷氏药学大全),16版,A.Osal,编辑(1980)中公开了这些技术。Active ingredients can also be encapsulated in microcapsules prepared, for example, by coascervation technology or interfacial polymerisation, for example in colloidal drug delivery systems (e.g. liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or microcapsules, respectively. Hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules in emulsion. These techniques are disclosed in Remington's Pharmaceutical Sciences, 16th Edition, A. Osal, ed. (1980).
也可应用经肺给药，例如使用吸入器或喷雾器，以及具有雾化剂的制剂。抗体也可以干粉组合物的形式施用入患者肺部，参阅例如US专利号6,514,496。Pulmonary administration, for example using an inhaler or nebulizer, and formulations with nebulizers may also be used. Antibodies can also be administered into the lungs of a patient in the form of dry powder compositions, see eg US Patent No. 6,514,496.
在一个具体实施方案中，也许希望在需要治疗的区域局部施用本发明之治疗抗体或组合物；这可通过以下方式实现：例如但不限于，局部输注、局部应用、注射、经由导管、以栓剂或植入的方式；所述植入物为多孔、非多孔或凝胶状物质，包括薄膜如sialastic膜或纤维。优选地，当施用本发明之抗体时，应注意采用蛋白不吸收或不吸附的材料。In a particular embodiment, it may be desirable to administer a therapeutic antibody or composition of the invention locally to the area in need of treatment; this may be accomplished by, for example and without limitation, local infusion, topical application, injection, via a catheter, with Suppository or means of implantation; said implants are porous, non-porous or gel-like substances, including thin films such as sialastic membranes or fibers. Preferably, when administering the antibodies of the present invention, care should be taken to use materials that do not absorb or adsorb proteins.
在另一实施方案中，该抗体可经由控制释放系统递送。在一个实施方案中，可使用泵(参阅Langer,Science 249:1527(1990)；Sefton,CRC Crit Ref Biomed Eng 14:201(1987)；Buchwald等人,Surgery 88:507(1980)；以及Saudek等人,N Engl J Med 321:574(1989))。在另一实施方案中，可使用聚合材料(参阅Medical Applications ofControlled Release,Langer 等人,编辑,CRC Press(1974)；Controlled DrugBioavailability,Drug Product Design and Performance,Smolen等人,eds.,Wiley(1984)；Ranger等人,J Macromol Sci Rev Macromol Chem 23:61(1983)；还参见Levy等人,Science 228:190(1985)；During等人,Ann Neurol 25:351(1989)；以及Howard等人,JNeurosurg 71:105(1989))。在又一实施方案中，控制释放系统可放在治疗靶的附近。In another embodiment, the antibody can be delivered via a controlled release system. In one embodiment, a pump can be used (see Langer, Science 249:1527 (1990); Sefton, CRC Crit Ref Biomed Eng 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al. People, N Engl J Med 321:574 (1989)). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer et al., eds., CRC Press (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen et al., eds., Wiley (1984) ; Ranger et al., J Macromol Sci Rev Macromol Chem 23:61 (1983); see also Levy et al., Science 228:190 (1985); During et al., Ann Neurol 25:351 (1989); and Howard et al., J Neurosurg 71:105 (1989)). In yet another embodiment, a controlled release system can be placed in the vicinity of the therapeutic target.
通过混合具有所需纯度的多肽与任选的"可药用的"载体、稀释剂、赋形剂或常用于本技术领域的稳定剂(即缓冲剂、稳定剂、防腐剂、等渗剂、非离子型洗涤剂、抗氧化剂以及其它多种添加剂)，参阅Remington's Pharmaceutical Sciences(雷氏药学大全),16th版，Osol,ed.(1980)，多肽或抗体治疗制剂可制备成冻干制剂或水溶液储存。这些添加剂在所用剂量和浓度下，对接受者一般都没有毒性，因此，赋形剂、稀释剂、载体等是可药用的。By mixing the polypeptide having the desired purity with optional "pharmaceutically acceptable" carriers, diluents, excipients or stabilizers commonly used in the art (i.e. buffers, stabilizers, preservatives, isotonic agents, non-ionic detergent, antioxidant and other various additives), refer to Remington's Pharmaceutical Sciences (Ray's Pharmaceutical Encyclopedia), 16th edition, Osol, ed. (1980), polypeptide or antibody therapeutic preparation can be prepared into lyophilized preparation or aqueous solution store. These additives are generally non-toxic to recipients at the dosages and concentrations employed, and therefore, excipients, diluents, carriers, etc. are pharmaceutically acceptable.
"分离"或"纯化"抗体基本上不含细胞物质，或来自细胞或组织来源或衍生蛋白的培养基的其它污染蛋白，或在化学合成的情况下基本上不含化学前体或其它化合物。"基本上不含细胞物质"这一措词包括抗体制品，其中多肽/蛋白与细胞的细胞组分分开，所述细胞的细胞组分中可以分离或重组产生所述抗体。因此，基本上不含细胞物质的抗体包括污染蛋白低于约30％、20％、10％、5％、2.5％或1％(干重)的抗体制品。当抗体是以重组方式产生时，也优选基本上不含培养基，即以蛋白制品体积计，培养基含量低于约20％、10％、5％、2.5％或1％。当抗体是以化学合成方式产生时，优选基本上不含化学前体或其它化学品和试剂，即目的抗体是与化学前体或涉及蛋白合成的其它化学品分开的。因此，这些抗体制品含有低于约30％、20％、10％、5％或1％(干重)的化学前体或目的抗体以外的其它化合物。在本发明一个优选的实施方案中，抗体是分离或纯化的。An "isolated" or "purified" antibody is substantially free of cellular material, or other contaminating proteins from the culture medium from which the cell or tissue source or protein is derived, or, in the case of chemical synthesis, chemical precursors or other compounds. The phrase "substantially free of cellular material" includes antibody preparations in which the polypeptide/protein is separated from the cellular components of the cells in which the antibody can be isolated or recombinantly produced. Thus, an antibody that is substantially free of cellular material includes antibody preparations that have less than about 30%, 20%, 10%, 5%, 2.5%, or 1% (dry weight) of contaminating protein. When the antibody is produced recombinantly, it is also preferably substantially free of medium, ie, contains less than about 20%, 10%, 5%, 2.5%, or 1% of medium by volume of the protein preparation. When the antibody is produced by chemical synthesis, it is preferably substantially free of chemical precursors or other chemicals and reagents, ie, the antibody of interest is separated from chemical precursors or other chemicals involved in protein synthesis. Accordingly, these antibody preparations contain less than about 30%, 20%, 10%, 5%, or 1% (dry weight) of chemical precursors or other compounds other than the antibody of interest. In a preferred embodiment of the invention, the antibodies are isolated or purified.
本文所用的术语"低至不可检测的聚集水平"，是指样品中含有不超过5％、不超过4％、不超过3％、不超过2％、不超过1％且往往不超过0.5％的聚集(按蛋白重量计)，例如以高效大小排阻层析法(HPSEC)测量。As used herein, the term "low to undetectable levels of aggregation" means that the sample contains no more than 5%, no more than 4%, no more than 3%, no more than 2%, no more than 1% and often no more than 0.5% Aggregation (by protein weight), for example measured by high performance size exclusion chromatography (HPSEC).
本文所用的术语"低至不可检测的片段化水平"，是指例如在HPSEC所确定的一个单峰处，或例如还原毛细管凝胶电泳(rCGE)所确定的两个(2)峰(重链和轻链)处，样品中含有等于或高于80％、85％、90％、95％、98％或99％总蛋白，且不含分别高于5％、高于4％、高于3％、高于2％、高于1％，或高于0.5％总蛋白的其它单峰。本文所用的rCGE是指还原条件下的毛细管凝胶电泳，该还原条件足以使抗体或抗体类型分子或抗体衍生分子中的二硫键被还原。The term "low to undetectable levels of fragmentation" as used herein refers to a single peak, e.g., as determined by HPSEC, or two (2) peaks, e.g., as determined by reducing capillary gel electrophoresis (rCGE). and light chains), the sample contains equal to or greater than 80%, 85%, 90%, 95%, 98% or 99% of total protein, and does not contain more than 5%, more than 4%, and more than 3 %, greater than 2%, greater than 1%, or other single peaks greater than 0.5% total protein. rCGE as used herein refers to capillary gel electrophoresis under reducing conditions sufficient to reduce disulfide bonds in antibodies or antibody-type molecules or antibody-derived molecules.
就含有IL-4和/或IL-13抗体或其结合片段的液体制剂而论，本文所用的术语"稳定"和"稳定的"是指制剂中的抗体或其抗原结合片段，在给定的制造、制备、运输和储存条件下，对于热和化学解折叠、聚集、降解或片段化的抵抗力。本发明的"稳定的"制剂在给定的制造、制备、运输和储存条件下，可保持等于或高于80％、85％、90％、95％、98％、99％或99.5％的生物活性。所述抗体制品的稳定性，可根据聚集、降解或片段化的程度来评估，并与参照进行比较；采用的方法是本技术领域内专业人员已知的，包括但不限于rCGE、十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)以及HPSEC。As used herein, the terms "stable" and "stable" in relation to liquid formulations containing IL-4 and/or IL-13 antibodies or binding fragments thereof mean that the antibodies or antigen-binding fragments thereof in the formulation are, at a given Resistance to thermal and chemical unfolding, aggregation, degradation or fragmentation under manufacturing, preparation, transport and storage conditions. "Stable" formulations of the invention retain equal to or greater than 80%, 85%, 90%, 95%, 98%, 99% or 99.5% of their biological active. The stability of the antibody preparation can be evaluated according to the degree of aggregation, degradation or fragmentation, and compared with the reference; the methods used are known to those skilled in the art, including but not limited to rCGE, dodecane Sodium sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and HPSEC.
术语"载体"是指与治疗剂一起施用的稀释剂、佐剂、赋形剂或运载体。这类生理载体可以是无菌液体，例如水和油，包括石油、动物、植物或合成来源的油，例如花生油、大豆油、矿物油、麻油等等。当药物组合物为静脉内施用时，水是适宜的载体。盐水溶液以及葡萄糖和甘油的水溶液也可作为液体载体使用，尤其是用于可注射溶液。适宜的药物赋形剂包括淀粉、葡萄糖、乳糖、蔗糖、明胶、麦芽、米(rice)、面粉、白垩、硅胶、硬脂酸钠、单硬脂酸甘油酯、滑石、氯化钠、脱脂奶粉、甘油、丙二醇、水、乙醇等等。如果需要，该组合物也可含有少量的润湿剂或乳化剂，或pH缓冲剂。该组合物可采取溶液剂、混悬剂、乳剂、片剂、丸剂、胶囊剂、散剂、缓释制剂、贮库(depot)等形式。该组合物可用传统的黏合剂和载体如甘油三酯配制成栓剂。口服制剂可含有标准载体，如药用级的甘露醇、乳糖、淀粉、硬脂酸镁、糖精钠(sodium saccharine)、纤维素、碳酸镁等。适宜载体的例子在"Remington'sPharmaceutical Sciences,Martin"中有所描述。这类组合物将含有效量的抗体，优选地纯化形式抗体，以及适量的载体以提供适合给患者施用的形式。如本领域内周知，该制剂将构建为适合于施用的方式。The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered. Such physiological carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a suitable carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, dry skim milk , Glycerin, Propylene Glycol, Water, Ethanol, etc. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The composition can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations, depots, and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulations can contain standard carriers, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. Examples of suitable carriers are described in "Remington's Pharmaceutical Sciences, Martin". Such compositions will contain an effective amount of the antibody, preferably in purified form, and an appropriate amount of carrier to provide it in a form suitable for administration to a patient. The formulations will be constructed in a manner suitable for administration as is known in the art.
缓冲液有助于将pH值维持在近似于生理条件的范围内。优选缓冲液存在的浓度范围为大约2mM到大约50mM。适宜用于本发明的缓冲液包括有机酸和无机酸及其盐，例如柠檬酸盐缓冲液(例如柠檬酸单钠-柠檬酸二钠混合物、柠檬酸-柠檬酸三钠混合物、柠檬酸-柠檬酸单钠混合物等)、琥珀酸盐缓冲液(例如琥珀酸-琥珀酸单钠混合物、琥珀酸-氢氧化钠混合物、琥珀酸-琥珀酸二钠混合物等)、酒石酸盐缓冲液(例如酒石酸-酒石酸钠混合物、酒石酸-酒石酸钾混合物、酒石酸-氢氧化钠混合物等)、富马酸盐缓冲液(例如富马酸-富马酸单钠混合物、富马酸-富马酸二钠混合物、富马酸单钠-富马酸二钠混合物等)、葡糖酸盐缓冲液(例如葡糖酸-葡糖酸钠混合物、葡糖酸-氢氧化钠混合物、葡糖酸-葡糖酸钾混合物等)、草酸盐缓冲液(例如草酸-草酸钠混合物、草酸-氢氧化钠混合物、草酸-草酸钾混合物等)、乳酸盐缓冲液(例如乳酸-乳酸钠混合物、乳酸-氢氧化钠混合物、乳酸-乳酸钾混合物等)以及乙酸盐缓冲液(例如乙酸-乙酸钠混合物、乙酸-氢氧化钠混合物等)。磷酸盐缓冲液、碳酸盐缓冲液、组氨酸缓冲液、三乙胺盐例如Tris、HEPES以及其它已知的此类缓冲液均可以使用。Buffers help maintain the pH in a range that approximates physiological conditions. Preferably, the buffer is present at a concentration ranging from about 2 mM to about 50 mM. Buffers suitable for use in the present invention include organic and inorganic acids and salts thereof, such as citrate buffers (e.g. monosodium citrate-disodium citrate mixture, citric acid-trisodium citrate mixture, citric acid-citric acid monosodium succinate mixture, etc.), succinate buffer (such as succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffer (such as tartaric acid- Sodium tartrate mixture, tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffer (such as fumaric acid-monosodium fumarate mixture, fumaric acid-disodium fumarate mixture, monosodium malate-disodium fumarate mixture, etc.), gluconate buffer (such as gluconate-sodium gluconate mixture, gluconate-sodium hydroxide mixture, gluconate-potassium gluconate mixture etc.), oxalate buffer (such as oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture, oxalic acid-potassium oxalate mixture, etc.), lactate buffer solution (such as lactic acid-sodium lactate mixture, lactic acid-sodium hydroxide mixture, lactic acid-potassium lactate mixture, etc.) and acetate buffer (such as acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). Phosphate buffer, carbonate buffer, histidine buffer, triethylamine salts such as Tris, HEPES and other known such buffers can be used.
可加入防腐剂以延迟微生物生长，加入量范围可为0.2％-1％(w/v)。适用于本发明的防腐剂包括苯酚、苯甲醇、间甲酚、对羟基苯甲酸甲酯、对羟基苯甲酸丙酯、十八烷基二甲基苄基氯化铵、benzyaconium卤化物(例如氯化物、溴化物和碘化物)、氯己双胺、对羟基苯甲酸烷基酯如对羟基苯甲酸甲酯或丙酯、儿茶酚、间苯二酚、环己醇以及3-戊醇。Preservatives may be added to retard microbial growth, and may range from 0.2% to 1% (w/v). Preservatives suitable for use in the present invention include phenol, benzyl alcohol, m-cresol, methylparaben, propylparaben, octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (such as chlorine compounds, bromides and iodides), hexamethylenediamine, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
等渗剂(isotonicifier)的存在可确保本发明之液体组合物的生理等渗性，其包括多元糖醇、优选地三元或三元以上的糖醇，例如甘油、赤藓醇、阿糖醇、木糖醇、山梨醇以及甘露醇。考虑到其它成分的相对量，多元醇可以约0.1％至约25％(重量)的量存在，优选地1％至5％。The presence of an isotonic agent (isotonicifier) can ensure the physiological isotonicity of the liquid composition of the present invention, which includes polysaccharide alcohols, preferably trivalent or higher sugar alcohols, such as glycerin, erythritol, arabitol , xylitol, sorbitol and mannitol. Given the relative amounts of other ingredients, the polyol may be present in an amount of from about 0.1% to about 25% by weight, preferably 1% to 5%.
稳定剂是指种类广泛的赋形剂，其功能范围可从填充剂至将治疗剂溶解或有助于预防变性或粘在容器壁上的添加剂。典型的稳定剂可以是多元糖醇；氨基酸，如精氨酸、赖氨酸、甘氨酸、谷氨酰胺、天冬酰胺、组氨酸、丙氨酸、鸟氨酸、L-亮氨酸、2-苯丙氨酸、谷氨酸、苏氨酸等；有机糖或糖醇，如乳糖、海藻糖、水苏糖、阿糖醇、赤藓醇、甘露醇、山梨醇、木糖醇、核糖醇、肌-肌醇(myoinisitol)、半乳糖醇、甘油等，包括环多醇如肌醇；聚乙二醇；氨基酸聚合物；含硫还原剂，如尿素、谷胱甘肽、硫辛酸、硫基乙酸钠、硫代甘油、α-单硫代甘油和硫代硫酸钠；低分子量多肽(即<10残基)；蛋白质，如人血清白蛋白、胎牛血清白蛋白、明胶或免疫球蛋白；亲水聚合物，如聚乙烯吡咯烷酮、糖类、单糖，如木糖、甘露糖、果糖、葡萄糖；二糖，如乳糖、麦芽糖和蔗糖；三糖，如棉子糖；多糖，如葡聚糖等等。稳定剂范围为每活性蛋白部分的0.1至10,000w/w。Stabilizers refer to a wide variety of excipients whose functions can range from fillers to additives that dissolve therapeutic agents or help prevent denaturation or sticking to container walls. Typical stabilizers can be polysaccharide alcohols; amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2 - Phenylalanine, glutamic acid, threonine, etc.; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol, ribose Alcohol, myo-inositol (myoinisitol), galactitol, glycerol, etc., including cyclic polyols such as inositol; polyethylene glycol; amino acid polymers; sulfur-containing reducing agents, such as urea, glutathione, lipoic acid, Sodium thioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weight polypeptides (ie, <10 residues); proteins, such as human serum albumin, fetal bovine serum albumin, gelatin, or immunoglobulins Proteins; hydrophilic polymers such as polyvinylpyrrolidone, sugars, monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose and sucrose; trisaccharides such as raffinose; polysaccharides such as Dextran and more. Stabilizers range from 0.1 to 10,000 w/w per active protein fraction.
其它多种赋形剂包括填充剂(如淀粉)、螯合剂(如EDTA)、抗氧化剂(如抗坏血酸、甲硫氨酸或维生素E)以及共溶剂。Various other excipients include fillers such as starch, chelating agents such as EDTA, antioxidants such as ascorbic acid, methionine or vitamin E, and co-solvents.
根据所治具体适应证的需要，本文的制剂也可包含一种以上活性化合物，优选地那些具有互补活性彼此不会不利影响的化合物。例如，也许希望进一步提供免疫抑制剂。这类化合物分子以有效地实现预期目的量组合存在。As required by the particular indication being treated, the formulations herein may also contain more than one active compound, preferably those compounds with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide immunosuppressants. Such compound molecules are present in combination in amounts effective to achieve the intended purpose.
本文所用的术语"表面活性剂"是指具有两亲结构的有机物，由相反溶解倾向的基团，通常是一个油溶性烃链和一个水溶性离子基团组成。表面活性剂可根据表面活性部分的电荷而划分为阴离子型、阳离子型和非离子型表面活性剂。表面活性剂经常作为润湿剂、乳化剂、增溶剂和分散剂，用于多种各样的药物组合物以及生物材料制品。The term "surfactant" as used herein refers to an organic substance with an amphiphilic structure consisting of groups with opposite solubility tendencies, usually an oil-soluble hydrocarbon chain and a water-soluble ionic group. Surfactants can be classified according to the charge of the surface active moiety as anionic, cationic and nonionic surfactants. Surfactants are frequently used as wetting, emulsifying, solubilizing and dispersing agents in a wide variety of pharmaceutical compositions as well as biomaterial preparations.
可加入非离子型表面活性剂或洗涤剂(又称为“润湿剂”)以助于治疗剂的溶解，并保护治疗蛋白以避免振荡引起的聚集，也使得制剂暴露于剪切力表面应激(shear surfacestress)而不会引起蛋白的变性。适宜的非离子型表面活性剂包括聚山梨酯(20、80等)、polyoxamer(184、188等)、多元醇以及聚氧乙烯脱水山梨糖醇单醚(等)。非离子型表面活性剂存在范围可为约0.05mg/ml至约1.0mg/ml，优选地约0.07mg/ml至约0.2mg/ml。Non-ionic surfactants or detergents (also known as "wetting agents") can be added to aid in the dissolution of the therapeutic agent, to protect the therapeutic protein from aggregation induced by shaking, and to expose the formulation to shear stress. Shock (shear surface stress) without causing protein denaturation. Suitable nonionic surfactants include polysorbate (20, 80, etc.), polyoxamer (184, 188, etc.), polyols and polyoxyethylene sorbitan monoether ( Wait). Nonionic surfactants may be present in the range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.
本文所用的术语"无机盐"是指任何因酸氢或酸中一部分或所有被金属或起金属作用的基团置换而生成的不含碳化合物，并在药物组合物和生物材料制剂中经常用作为张力调节化合物。最常见的无机盐是NaCl、KCl、NaH2PO4等。As used herein, the term "inorganic salt" refers to any carbon-free compound formed by the hydrogen acid or some or all of the acid being replaced by a metal or a metal-acting group, and is often used in pharmaceutical compositions and biological material preparations. As a tonicity-modulating compound. The most common inorganic salts are NaCl, KCl, NaH2 PO4 and so on.
本发明包括在医生诊所或实验室的商业冰箱和冷冻室温度(如约-20℃至约5℃)下具有稳定性的液体制剂，上述稳定性是用例如高效大小排阻层析法(HPSEC)来评估的，用于储存目的，例如约60天、约120天、约180天、约1年、约2年或以上。本发明的液体制剂在室温下也显示了至少多个小时的稳定性，例如根据HSPEC评估的，例如在使用之前1小时、2小时或约3小时。The present invention includes liquid formulations that are stable at commercial refrigerator and freezer temperatures (e.g., about -20°C to about 5°C) in a physician's office or laboratory, as determined by, for example, high performance size exclusion chromatography (HPSEC) For storage purposes, for example, about 60 days, about 120 days, about 180 days, about 1 year, about 2 years or more. The liquid formulations of the invention also exhibit stability at room temperature for at least a number of hours, eg as assessed by HSPEC, eg 1 hour, 2 hours or about 3 hours prior to use.
术语"小分子"和类似术语包括但不限于肽、素拟肽(peptidomimetics)、氨基酸、氨基酸类似物、多核苷酸、多核苷酸类似物、核苷酸、核苷酸类似物、分子量为每摩尔小于约10,000克的有机或无机化合物(即包括杂有机和/有机金属化合物)、分子量为每摩尔小于约5,000克的有机或无机化合物、分子量为每摩尔小于约1,000克的有机或无机化合物、分子量为每摩尔小于约500克的有机或无机化合物，以及这类化合物的盐、酯及其它可药用的形式。The term "small molecule" and like terms include, but are not limited to, peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, nucleotides, nucleotide analogs, molecular weights per Organic or inorganic compounds having a molar weight of less than about 10,000 grams (i.e. including heteroorganic and/or organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, Organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters and other pharmaceutically acceptable forms of such compounds.
因此，在癌症的情况下，本发明的抗体可以单独施用，也可与其它类型的癌症治疗组合施用，包括传统的化疗剂(紫杉醇(paclitaxel)、卡铂(carboplatin)、顺铂(cisplatin)和多柔比星(doxorubicin))，抗EGFR剂(吉非替尼(gefitinib)、埃罗替尼(erlotinib)和西妥昔单抗(cetuximab))，抗血管生成剂(贝伐单抗(bevacizumab)和舒尼替尼(sunitinib))，以及免疫调节剂如干扰素α和沙利度胺(thalidomide)。Thus, in the case of cancer, the antibodies of the invention can be administered alone or in combination with other types of cancer treatments, including traditional chemotherapeutic agents (paclitaxel, carboplatin, cisplatin, and Doxorubicin), anti-EGFR agents (gefitinib, erlotinib, and cetuximab), anti-angiogenic agents (bevacizumab ) and sunitinib), and immunomodulators such as interferon alpha and thalidomide.
本文所用的术语"治疗剂"是指可用于与异常IL-4和/或IL-13代谢和活性相关的疾病、障碍、病症等的治疗、管理或改善的任何活性剂。The term "therapeutic agent" as used herein refers to any active agent useful for the treatment, management or amelioration of a disease, disorder, condition, etc. associated with abnormal IL-4 and/or IL-13 metabolism and activity.
此外，本发明的抗体可以与多种效应分子缀合，例如异源的多肽、药物、放射性核苷酸或毒素，参阅例如WO 92/08495；WO 91/14438；WO 89/12624；US专利号5,314,995；以及EPO 396,387。抗体及其片段可与治疗部分缀合，例如细胞毒素(例如细胞抑制剂或杀细胞剂)、治疗剂或放射性金属离子(例如α发射体如213Bi)。细胞毒素或细胞毒性剂包括任何损害细胞的活性剂。例子包括紫杉醇(paclitaxol)、松胞菌素B(cytochalasin B)、短杆菌肽D(gramicidin D)、溴乙啶(ethidium bromide)、依米丁(emetine)、丝裂霉素(mitomycin)、依托泊苷(etoposide)、替尼泊苷(tenoposide)、长春新碱(vincristine)、长春碱(vinblastine)、秋水仙碱(colchicine)、多柔比星(doxorubicin)、柔红霉素(daunorubicin)、二羟基蒽醌(dihydroxy anthracindione)、米托蒽醌(mitoxantrone)、米拉霉素(mithramycin)、放线菌素(actinomycin D)、1-去氢睾酮(1-dehydrotestosterone)、糖皮质激素(glucocorticoids)、普鲁卡因(procaine)、丁卡因(tetracaine)、利多卡因(lidocaine)、普萘洛尔(propranolol)和嘌呤霉素(puromycin)及其类似物或同源物。治疗剂包括但不限于抗代谢物(例如甲氨蝶呤(methotrexate)、6-巯基嘌呤(6-mercaptopurine)、6-硫代鸟嘌呤(6-thioguanine)、阿糖胞苷(cytarabine)、5-氟脲嘧啶(5-fluorouracil)和氮烯咪胺(decarbazine))，烷基化剂(例如氮芥(mechlorethamine)、苯丁酸氮芥(chlorambucil)、美法仑(melphalan)、卡莫司汀(carmustine(BSNU))和洛莫司汀(lomustine(CCNU))、环磷酰胺(cyclophosphamide)、白消安(busulfan)、二溴甘露醇(dibromomannitol)、链脲霉素(streptozotocin)、丝裂霉素C(mitomycin C)和顺二氯二胺铂(II)(DDP)顺铂(cisplatin))，蒽环类(anthracyclines)(例如柔红霉素(daunorubicin)、道诺霉素(daunomycin)和多柔比星(doxorubicin))，抗生素(例如更生霉素(dactinomycin)、放线菌素(actinomycin)、博来霉素(bleomycin)、米拉霉素(mithramycin)和氨茴霉素(anthramycin(AMC)))，以及抗有丝分裂剂(例如长春新碱(vincristine)和长春碱(vinblastine))。In addition, the antibodies of the invention can be conjugated to various effector molecules, such as heterologous polypeptides, drugs, radionucleotides or toxins, see for example WO 92/08495; WO 91/14438; WO 89/12624; US Patent No. 5,314,995; and EPO 396,387. Antibodies and fragments thereof can be conjugated to therapeutic moieties, such as cytotoxins (eg, cytostatic or cytocidal agents), therapeutic agents, or radioactive metal ions (eg, alpha emitters such as213 Bi). A cytotoxin or cytotoxic agent includes any agent that damages cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, ethidium Etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, Dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids ), procaine, tetracaine, lidocaine, propranolol and puromycin and their analogs or congeners. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5 - 5-fluorouracil (5-fluorouracil and decarbazine), alkylating agents (eg, mechlorethamine, chlorambucil, melphalan, carlimus carmustine (BSNU) and lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, silk Mitomycin C and cisplatin (DDP) cisplatin), anthracyclines (eg daunorubicin, daunomycin and doxorubicin), antibiotics (such as dactinomycin, actinomycin, bleomycin, mithramycin, and anthramycin (AMC))), and antimitotic agents (eg vincristine and vinblastine).
使此类治疗部分与抗体缀合的技术是众所周知的，参阅例如Arnon等人,inMonoclonal Antibodies and Cancer Therapy,Reisfeld等人(eds.),p.243-56AlanR.Liss(1985)；Hellstrom等人,in Controlled Drug Delivery,2nd ed.,Robinson等人,eds.,p.623-53,Marcel Dekker(1987)；Thorpe,in Monoclonal Antibodies'84:Biological And Clinical Applications,Pinchera等人,eds.,p.475-506(1985)；Monoclonal Antibodies For Cancer Detection and Therapy,Baldwin等人,eds.,p.303-16,Academic Press(1985)；和Thorpe等人,Immunol Rev 62:119(1982)。可选地，抗体可以与第二种抗体缀合，以形成抗体异缀合物，如双功能抗体，参阅例如US专利号4,676,980。Techniques for conjugating such therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., in Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), p. 243-56 Alan R. Liss (1985); Hellstrom et al., in Controlled Drug Delivery, 2nd ed., Robinson et al., eds., p.623-53, Marcel Dekker (1987); Thorpe, in Monoclonal Antibodies'84: Biological And Clinical Applications, Pinchera et al., eds., p. 475-506 (1985); Monoclonal Antibodies For Cancer Detection and Therapy, Baldwin et al., eds., p. 303-16, Academic Press (1985); and Thorpe et al., Immunol Rev 62:119 (1982). Alternatively, an antibody can be conjugated to a second antibody to form an antibody heteroconjugate, such as a diabody, see eg US Pat. No. 4,676,980.
本发明的缀合物可用于改变给定的生物反应，治疗剂或药物部分不应被理解为只限于传统的化学治疗剂。例如，该药物部分可以是具有所需生物活性的蛋白或多肽。这类蛋白可包括，例如毒素，如相思豆毒蛋白、蓖麻毒蛋白A、假单胞菌外毒素，或白喉毒素；蛋白，如肿瘤坏死因子、α-干扰素、β-干扰素、神经生长因子、血小板衍生生长因子、组织纤溶酶原激活物；凋亡剂，例如TNF-α、TNF-β、AIM I(WO 97/33899)、AIM II(WO 97/34911)、Fas配体(Takahashi等人,Int Immunol,6:1567(1994)),VEGF(WO 99/23105)；血栓形成剂；抗血管生成剂，例如血管他丁或内皮他丁；或生物反应改性剂，例如淋巴因子、白细胞介素-1(IL-1)、白细胞介素-2(IL-2)、白细胞介素-6(IL-6)，粒细胞巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(GCSF)或其它生长因子。The use of the conjugates of the present invention to alter a given biological response, therapeutic agent or drug moiety should not be construed as limited to traditional chemotherapeutic agents. For example, the drug moiety can be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; proteins such as tumor necrosis factor, alpha-interferon, beta-interferon, neuronal Growth factors, platelet-derived growth factor, tissue plasminogen activator; apoptotic agents such as TNF-alpha, TNF-beta, AIM I (WO 97/33899), AIM II (WO 97/34911), Fas ligand (Takahashi et al., Int Immunol, 6:1567 (1994)), VEGF (WO 99/23105); thrombogenic agents; anti-angiogenic agents, such as angiostatin or endostatin; or biological response modifiers, such as Lymphokines, interleukin-1 (IL-1), interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (GCSF) or other growth factors.
用于体内施用的制剂必须是无菌的。这可通过例如无菌过滤膜过滤而实现。例如，本发明的液体制剂可通过用0.2μm或0.22μm过滤器过滤而灭菌。Preparations for in vivo administration must be sterile. This can be achieved, for example, by filtration through sterile filtration membranes. For example, liquid formulations of the present invention can be sterilized by filtration through a 0.2 μm or 0.22 μm filter.
可以制备缓释型制品。缓释型制品的适宜例子包括含有抗体的固体疏水聚合物的半渗透基质，该基质以成形物品的形式，例如薄膜或基质。缓释型基质的例子包括聚酯、水凝胶(例如聚(2-羟乙基甲基丙烯酸酯)、聚(乙烯醇)、聚丙交酯(US专利号3,773,919)、L-谷氨酸和乙基-L-谷氨酸酯的共聚物、不可降解的乙烯-醋酸乙烯酯、可降解的乳酸-乙醇酸共聚物(例如由乳酸-乙醇酸共聚物构成的可注射微球)以及聚-D-(-)-3-羟基丁酸。例如乙烯-醋酸乙烯酯和乳酸-乙醇酸等的聚合物能够释放分子长达100天以上，但某些水凝胶释放蛋白的时间则较短。取决于所涉及的机制，可以制订出用于稳定的合理策略。例如，如果发现聚集机制是通过硫代-二硫化物交换而形成分子间的S-S键，那么通过修饰硫氢基残基、从酸性溶液冷冻干燥、控制含湿量、使用适当的添加剂、氨基酸取代和开发特异的聚合物基质组合物就可以达到稳定。Sustained release formulations can be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody in the form of shaped articles such as films or matrices. Examples of sustained release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl methacrylate), poly(vinyl alcohol), polylactide (US Patent No. 3,773,919), L-glutamic acid and Ethyl-L-glutamate copolymers, non-degradable ethylene-vinyl acetate, degradable lactic-co-glycolic acid (such as injectable microspheres composed of lactic-co-glycolic acid), and poly- D-(-)-3-Hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic-glycolic acid can release molecules for longer than 100 days, but certain hydrogels release proteins for a shorter period of time. Depending on the mechanism involved, rational strategies for stabilization can be worked out. For example, if the mechanism of aggregation is found to be intermolecular S-S bond formation by thio-disulfide exchange, then by modifying sulfhydryl residues, from Stabilization can be achieved by lyophilization of acidic solutions, control of moisture content, use of appropriate additives, amino acid substitutions and development of specific polymer matrix compositions.
抗体或变体组合物将以符合优良医疗实践的方式配制、定量以及施用。这方面考虑的因素包括要治的具体障碍、要治的具体哺乳动物或人、个体患者的临床状况、致病原因、递送活性剂部位、给药方法、给药方案以及医疗工作者所知的其它因素。要被施用的抗体或变体的"治疗有效量"将取决于这些考虑，并可以是为了预防、改善或治疗某种IL-4和/或IL-13介导的疾病、状况或障碍所必须的最低量。Antibody or variant compositions will be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal or human being being treated, the individual patient's clinical condition, etiology, site of delivery of the active agent, method of administration, regimen, and knowledge of the medical practitioner. other factors. The "therapeutically effective amount" of the antibody or variant to be administered will depend on these considerations and may be necessary for the prevention, amelioration or treatment of a certain IL-4 and/or IL-13 mediated disease, condition or disorder minimum amount.
任选地，抗体或变体与目前用于预防或治疗所讨论障碍的一种或多种活性剂一起配制。此类其它活性剂的有效量取决于制剂中存在的抗体的量、障碍或治疗的类型，以及上面讨论的其它因素。这些通常与此前所用的剂量一样且通过同样的给药途径使用，或为此前所用剂量的约从1至99％。Optionally, the antibody or variant is formulated with one or more active agents currently used in the prevention or treatment of the disorder in question. The effective amount of such other active agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are usually the same doses and by the same route of administration as previously used, or about from 1 to 99% of the previously used doses.
此处所使用的术语"有效量"系指下述的疗法(如预防或治疗活性剂)的量，所述量足以降低IL-4和/或IL-13介导的疾病之严重性和/或缩短其持续时间，改善其一种或多种症状，防止IL-4和/或IL-13介导的疾病的发展或引起疾病的退化，或足以防止IL-4和/或IL-13介导的疾病或其一种或多种症状的发展、复发、发作或进展，或加强或改善另一种对治疗IL-4和/或IL-13介导的疾病有用的疗法(如另一种治疗活性剂)的预防和/或治疗效果。As used herein, the term "effective amount" refers to an amount of a therapeutic (e.g. prophylactic or therapeutic active agent) sufficient to reduce the severity of IL-4 and/or IL-13 mediated disease and/or shorten its duration, ameliorate one or more of its symptoms, prevent the development of IL-4 and/or IL-13-mediated disease or cause regression of the disease, or be sufficient to prevent IL-4 and/or IL-13-mediated development, relapse, onset or progression of the disease or one or more of its symptoms, or to augment or ameliorate another therapy useful for treating an IL-4 and/or IL-13 mediated disease (such as another treatment prophylactic and/or therapeutic effects of active agents).
在特定疾病或状况的使用或治疗中有效的治疗抗体或其片段的量将取决于该疾病或状况的性质，并可通过标准的临床技术测定。当可能时，可先在体外得出剂量－反应曲线以及本发明的药物组合物。如果适宜的动物模型系统可用，同样可获得剂量－反应曲线，并利用其外推出实践本技术领域已知的方法的适宜的人剂量。但是，基于本领域的常识，有效地促进降低炎症作用的药物组合物，例如，可提供的局部治疗剂浓度为约5至20ng/ml之间，优选地约10至20ng/ml之间。The amount of therapeutic antibody or fragment thereof effective in the use or treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. When possible, dose-response curves and pharmaceutical compositions of the present invention can first be obtained in vitro. If an appropriate animal model system is available, dose-response curves can likewise be obtained and used to extrapolate appropriate human doses for practicing methods known in the art. However, based on common knowledge in the art, a pharmaceutical composition effective to promote the reduction of inflammation, for example, may provide a topical therapeutic agent concentration between about 5 and 20 ng/ml, preferably between about 10 and 20 ng/ml.
在优选的实施方案中，治疗用多肽、抗体或其片段的水溶液可以皮下注射施用。每次剂量的范围按每千克重量计可为约0.5mg至约50mg，或更优选地，按每千克体重计为约3mg至约30mg。针对具体的疾病、患者群体、施用方式等，可凭经验确定该剂量，以实践本技术领域内已知的药学方法。In a preferred embodiment, aqueous solutions of therapeutic polypeptides, antibodies or fragments thereof may be administered by subcutaneous injection. Each dose may range from about 0.5 mg to about 50 mg per kilogram of body weight, or more preferably, from about 3 mg to about 30 mg per kilogram of body weight. For a particular disease, patient population, mode of administration, etc., the dose can be determined empirically by practicing pharmaceutical methods known in the art.
取决于许多临床因素，包括疾病类型、疾病严重程度以及患者对治疗剂的敏感度，皮下注射的剂量安排可在每周一次至每天一次的范围内变化。The dosing schedule for subcutaneous injections can vary from weekly to daily depending on a number of clinical factors, including disease type, disease severity, and patient sensitivity to the therapeutic agent.
本发明提供了制备抗体或其IL-4和/或IL-13结合片段的液体制剂的方法，其包括将纯化抗体的级分浓缩至约15mg/ml、约20mg/ml、约30mg/ml、约40mg/ml、约50mg/ml、约60mg/ml、约70mg/ml、约80mg/ml、约90mg/ml、约100mg/ml、约200mg/ml、约250mg/ml、约300mg/ml或更高的最终浓度，例如使用具有适当分子量(mw)截断值(cutoff)(例如对于其F(ab')2片段的截断值为30KD；对于Fab片段的截断值为10KD)的半渗透膜。The invention provides a method of preparing a liquid formulation of an antibody or IL-4 and/or IL-13 binding fragment thereof comprising concentrating fractions of the purified antibody to about 15 mg/ml, about 20 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, about 90 mg/ml, about 100 mg/ml, about 200 mg/ml, about 250 mg/ml, about 300 mg/ml, or Higher final concentrations, e.g. using a semi-permeable medium with an appropriate molecular weight (mw) cutoff (e.g. 30KD for its F(ab')2 fragment;10KD for itsFab fragment) membrane.
此外，本发明也包括了稳定的、具有改善体内半衰期的目的产品的液体制剂。因此，目的抗体在受试者(优选地人)体内的半衰期为3天以上、7天以上、10天以上、15天以上、25天以上、30天以上、35天以上、40天以上、45天以上、2月以上、3月以上、4月以上、5月以上或更长。Furthermore, the present invention also encompasses stable liquid formulations of the desired product with improved in vivo half-life. Therefore, the half-life of the antibody of interest in a subject (preferably a human) is 3 days or more, 7 days or more, 10 days or more, 15 days or more, 25 days or more, 30 days or more, 35 days or more, 40 days or more, 45 days or more. More than days, more than 2 months, more than 3 months, more than 4 months, more than 5 months or longer.
为了延长抗体在体内的血清循环，可采用多种各样的技术。例如，惰性聚合物分子如高分子量聚乙二醇(PEG)，可以通过PEG的位点特异性缀合到抗体的N端或C端或通过赖氨酸残基上存在的ε氨基连接到抗体上，其中可以用或不用多功能连接体。可以采用导致生物活性损失最小的线性或支链聚合物衍生。缀合程度可通过SDS-PAGE和质谱密切监测，以确保PEG分子与抗体的妥善缀合。未反应的PEG可通过大小排阻或离子交换层析与抗体-PEG缀合物分离。采用本领域专业人员已知的方法，例如本文所述的免疫测定法，可以测定PEG衍生抗体的结合活性以及体内功效。To prolong serum circulation of antibodies in vivo, a variety of techniques can be employed. For example, an inert polymer molecule such as high molecular weight polyethylene glycol (PEG), can be site-specifically conjugated to the N- or C-terminus of the antibody via PEG or attached to the antibody via the epsilon amino group present on a lysine residue. on, with or without a multifunctional linker. Derivatization with linear or branched polymers can be employed resulting in minimal loss of biological activity. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of the PEG molecule to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or ion exchange chromatography. The binding activity and in vivo efficacy of PEG-derived antibodies can be determined using methods known to those skilled in the art, such as the immunoassays described herein.
在体内具有增加的半衰期的抗体，也可通过将一个或多个氨基酸修饰(即取代、插入或缺失)引入IgG恒定结构域或其FcR结合片段(如Fe或铰链Fe结构域片段)来产生，参阅例如WO 98/23289；WO 97/34631；以及US专利号6,277,375。Antibodies with increased half-life in vivo can also be achieved by introducing one or more amino acid modifications (i.e., substitutions, insertions, or deletions) into the IgG constant domain or itsFcR -binding fragment (such as anFe or hingeFe domain fragment). ) to produce, see eg WO 98/23289; WO 97/34631; and US Patent No. 6,277,375.
此外，抗体可与白蛋白缀合使得抗体在体内更稳定，或在体内具有较长的半衰期。这些技术是本领域内众所周知的，参阅例如WO 93/15199、WO 93/15200和WO 01/77137；以及EPO 413,622。抗体也可通过例如糖基化、乙酰化、磷酸化、酰胺化、用已知保护基/阻断基衍生化、蛋白水解切割、与细胞配体或其它蛋白连接等来修饰。In addition, the antibody can be conjugated to albumin to make the antibody more stable in vivo, or have a longer half-life in vivo. These techniques are well known in the art, see eg WO 93/15199, WO 93/15200 and WO 01/77137; and EPO 413,622. Antibodies can also be modified by, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like.
在一项实施方案中，按照常规步骤将该组合物配制成适合于人静脉内给药的药物组合物。通常，静脉内给药的组合物是在无菌等渗缓冲水溶液中的溶液。在必要的情形，该组合物也可包含增溶剂和局部麻醉剂如利多卡因或其它"卡因"类麻醉剂，以减轻注射部位的疼痛。通常，多种成分是以单位剂量的形式单独供应或混合在一起供应，例如作为在密封容器中的干燥冻干粉末或无水浓缩物，所述密封容器如安瓿瓶或囊，并标明活性成分的量。当组合物以输注方式给药时，其可被分散在含有无菌药用级水或盐水的输注瓶中。当组合物以注射方式给药时，可提供无菌注射用水或盐水的安瓿瓶，例如以试剂盒形式提供，使得成分可在施用前先混合。In one embodiment, the composition is formulated into a pharmaceutical composition suitable for intravenous administration to humans according to conventional procedures. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also contain solubilizers and local anesthetics such as lidocaine or other "caine" anesthetics to relieve pain at the injection site. Typically, the ingredients are supplied individually or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, and labeled with the active ingredient amount. When the composition is administered by infusion, it may be dispensed in an infusion bottle containing sterile pharmaceutical grade water or saline. When the composition is administered by injection, ampoules of sterile water for injection or saline can be provided, eg, in kit form, so that the ingredients can be mixed prior to administration.
本发明也提供，本发明的液体制剂是装在密封容器中，如安瓿瓶或囊(sachet)中，其上标明目的产品的量。本发明的液体制剂可在标明抗体或抗体片段的量和浓度的密封容器中。例如，本发明的液体制剂可在密封容器中供应，其具有的IL-4和/或IL-13抗体至少为15mg/ml、20mg/ml、30mg/ml、40mg/ml、50mg/ml、60mg/ml、70mg/ml、80mg/ml、90mg/ml、100mg/ml、150mg/ml、200mg/ml、250mg/ml，或300mg/ml，以1ml、2ml、3ml、4ml、5ml、6ml、7ml、8ml、9ml、10ml、15ml或20ml的量。The present invention also provides that the liquid formulation of the present invention is contained in a sealed container, such as an ampoule or a sachet, on which the amount of the desired product is indicated. Liquid formulations of the invention may be in sealed containers indicating the amount and concentration of the antibody or antibody fragment. For example, a liquid formulation of the invention may be supplied in a sealed container having at least 15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, 60 mg of IL-4 and/or IL-13 antibody 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml , 8ml, 9ml, 10ml, 15ml or 20ml.
提供了含有对上述障碍治疗有用物质的制备物品。该制备物品包括容器和标签。适宜的容器包括例如瓶子、小瓶、注射器和试管。容器可从多种材料形成，例如玻璃或塑料。容器容纳能有效地诊断、预防或治疗IL-4和/或IL-13介导的状况或疾病的组合物，并可具有无菌入口(例如容器可以是静脉内溶液袋或小瓶，其具有能被皮下注射针头刺破的塞子)。容器上的标签或附带的标签标明该组合物是用于治疗所选的状况。该制备物品还可包括第二个容器，其含有可药用的缓冲液，如磷酸缓冲盐溶液、Ringer's溶液和葡萄糖溶液。它还可包括从商业和用户的立场看来所需的其它材料，包括缓冲液、稀释剂、过滤器、针头、注射器以及使用说明的药品说明书。Articles of manufacture containing substances useful in the treatment of the aforementioned disorders are provided. The article of manufacture includes a container and a label. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers can be formed from a variety of materials, such as glass or plastic. The container contains a composition effective for diagnosing, preventing, or treating an IL-4 and/or IL-13 mediated condition or disease, and may have a sterile inlet (e.g., the container may be an intravenous solution bag or vial, which has an A stopper punctured by a hypodermic needle). The label on or accompanying the container indicates that the composition is used to treat the condition of choice. The article of manufacture may also include a second container containing a pharmaceutically acceptable buffer, such as phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including buffers, diluents, filters, needles, syringes, and package inserts for instructions for use.
为了技术人员有利，本发明将以下列非限制性实例示例说明，所述实例描述了一些本发明可籍以实施或在其中本发明可以实施的实施方案。For the benefit of the skilled person, the invention will be illustrated by the following non-limiting examples, which describe some of the embodiments by which or in which the invention may be practiced.
实施例Example
实施例1:小鼠抗人IL-13单克隆抗体克隆B-B13的Fv结构域的测序Embodiment 1: the sequencing of the Fv domain of mouse anti-human IL-13 monoclonal antibody clone B-B13
下面的方法中所使用的试剂为购自Cell Sciences,Inc.(Canton,MA,USA)的小鼠抗IL-13单克隆抗体克隆B-B13。Cell Sciences为抗体B-B13的制备商Diaclone(France)的美国分销商。The reagent used in the following method was mouse anti-IL-13 monoclonal antibody clone B-B13 purchased from Cell Sciences, Inc. (Canton, MA, USA). Cell Sciences is the manufacturer of antibody B-B13 Diaclone ( France) US distributor.
抗IL-13单克隆抗体克隆B-B13的氨基酸序列是利用Edman N端测序技术和质谱分析的组合测定的。抗体经过下述不同方法的处理，以便产生多肽或肽片段，这些片段然后用不同的方法分级分离以便制备样品用于后面的Edman N端测序和液相色谱/质谱/质谱(LC-MS/MS)分析，这是用相关的蛋白质序列数据库肽匹配。The amino acid sequence of anti-IL-13 monoclonal antibody clone B-B13 was determined using a combination of Edman N-terminal sequencing and mass spectrometry. Antibodies are processed in various ways as described below to produce polypeptides or peptide fragments which are then fractionated in various ways to prepare samples for subsequent Edman N-terminal sequencing and liquid chromatography/mass spectrometry/mass spectrometry (LC-MS/MS ) analysis, which is matched against peptides in related protein sequence databases.
抗体的SDS-Page，经过或不经过焦谷氨基肽酶(pyrogluamino)处理以分离重链和轻链，然后印迹到聚偏氟乙烯(PVDF)膜和并进行条带的Edman N端测序。SDS-Page of antibodies, with or without pyrogluamino-treatment to separate heavy and light chains, followed by blotting onto polyvinylidene fluoride (PVDF) membranes and Edman N-terminal sequencing of bands.
用抗体的特异蛋白酶有限地部分水解，接着进行SDS-Page并印迹到PVDF膜和并进行条带的Edman N端测序。Limited partial hydrolysis with antibody-specific proteases followed by SDS-Page and blotting onto PVDF membranes and Edman N-terminal sequencing of the bands.
对整个抗体或重链和轻链SDS-Page凝胶条带进行有限部分化学切割，接着进行SDS-Page并印迹到PVDF膜和并进行条带的Edman N端测序。Limited fraction chemical cleavage of whole antibody or heavy and light chain SDS-Page gel bands followed by SDS-Page and blot to PVDF membrane and Edman N-terminal sequencing of the bands.
对整个抗体或重链和轻链的SDS-Page凝胶条带以特异蛋白酶进行蛋白水解并进行LC/MS/MS分析。SDS-Page gel bands of the whole antibody or heavy and light chains were proteolyzed with specific proteases and analyzed by LC/MS/MS.
将重链和轻链的SDS-Page凝胶条带以特异蛋白酶进行蛋白水解，接着用反向高压色谱分级分离(rp-hplc)，再进行各级分的Edman N端测序和LC/MS/MS分析。The SDS-Page gel bands of the heavy and light chains were proteolyzed with specific proteases, followed by fractionation by reverse high-pressure chromatography (rp-hplc), and then Edman N-terminal sequencing and LC/MS/ MS analysis.
以木瓜蛋白酶对抗体作有限的蛋白水解，通过SDS-Page分级分离Fd(抗体重链VH-CH1片段)凝胶条带，以特异蛋白酶蛋白水解，反向高效液相色谱(rp-hplc)，再进行各级分的Edman N端测序和LC/MS/MS分析。Limited proteolysis of antibody with papain, fractionation of Fd (antibody heavy chain VH-CH1 fragment) gel band by SDS-Page, proteolysis with specific protease, reverse high performance liquid chromatography (rp-hplc), Edman N-terminal sequencing and LC/MS/MS analysis of each fraction were performed.
实施例2:小鼠抗人IL-4单克隆抗体克隆8D4-8的Fv结构域测序Example 2: Fv domain sequencing of mouse anti-human IL-4 monoclonal antibody clone 8D4-8
试剂小鼠抗IL-4单克隆抗体克隆8D4-8购自Biozol diagnostica Vertrieb GmbH(Eching,德国)。Biozol为生产8D4-8抗体的BioLegend(San Diego,CA,USA)之德国分销商。Reagents Mouse anti-IL-4 monoclonal antibody clone 8D4-8 was purchased from Biozol diagnostica Vertrieb GmbH (Eching, Germany). Biozol is the German distributor of BioLegend (San Diego, CA, USA), which produces the 8D4-8 antibody.
小鼠抗IL-4单克隆抗体(克隆8D4-8)的氨基酸序列是利用Edman测序和质谱分析组合测定的(Pham等人,2006,Anal.Biochem.352:77-86；Roberts等人,2005,Anal.Chem.67:3613-25)。简言之，抗体被首先分离成轻链和重链，然后每个链用序列特异的蛋白酶或化学方法切割。得到的肽用反向色谱分离并用基质辅助激光解吸/电离质谱(MALDI)和/或LC-MS/MS分析。独特的肽以及完整的重链和轻链然后进行Edman测序，以准确地测定蛋白质的序列。The amino acid sequence of the mouse anti-IL-4 monoclonal antibody (clone 8D4-8) was determined using a combination of Edman sequencing and mass spectrometry (Pham et al., 2006, Anal. Biochem. 352:77-86; Roberts et al., 2005 , Anal. Chem. 67:3613-25). Briefly, antibodies are first separated into light and heavy chains, and then each chain is cleaved with sequence-specific proteases or chemicals. The resulting peptides were separated by reverse phase chromatography and analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI) and/or LC-MS/MS. The unique peptides as well as the complete heavy and light chains are then subjected to Edman sequencing to accurately determine the sequence of the protein.
实施例3:小鼠抗人IL-13单克隆抗体克隆B-B13的Fv结构域的人源化Example 3: Humanization of the Fv domain of mouse anti-human IL-13 monoclonal antibody clone B-B13
此处所描述的人源化规程被用来实现B-B13克隆的人源化。建议六个人源化的形式，包括CDR中的突变来解决有问题的残基(脱酰胺位点、暴露于溶剂的甲硫氨酸、酸不稳定位置)。The humanization protocol described here was used to achieve humanization of the B-B13 clone. Six humanized versions were suggested, including mutations in the CDRs to address problematic residues (deamidation sites, solvent-exposed methionines, acid-labile positions).
B-B13的VL&VH序列与2007年7月的蛋白质数据库(PDB)版本进行blast。检索出大多数类似的轻链和重链氨基酸序列。发现可变轻链中最同源的为1EGJ。发现可变重链中最同源的为1FNS。1EGJ&1FNS结构被用来建造可变结构域的同源性模型，然后利用分子操作环境(MOE)中实施的标准程序对其进行能量最小化。MOE是由Chemical Computing group提供的一套综合性的计算机辅助药物设计软件。其后对B-B13的3D同源性模型在广义波恩隐含溶剂(Generalized Born implicit solvent)中进行了1.7纳秒的分子动力学(MD)计算。然后对每一个B-B13氨基酸，利用得到的1,700个MD轨迹快照，与参照medoid位置相比较，计算其均方根差(rmsd)的分布。最后用一个统计试验对每个氨基酸的均方根差分布与全局均方根差分布进行比较，以决定该氨基酸从MD计算来看，是否有足够的柔性而有可能被视为与B细胞受体相互作用，从而成为免疫反应活化的原因。将鼠B-B13可变区的柔性位置与下载到本地的2007年1月版ImMunoGeneTics数据库中人抗体序列对应的位置进行了比较。只有那些柔性大于平均值三倍的残基以及一些保存了这些柔性残基3D结构的侧翼残基才被保留用于搜索。选择含相同柔性残基最多的人抗体可变区，特别考虑位于CDR的范围内的位置，以便替换鼠B-B13抗体可变区的柔性残基。CDR中的一些突变也包括在提议的形式中以避免有问题的残基。研究了下面的序列基序：Asp-Pro(酸性不稳定键)、Asn-X-Ser/Thr(糖基化)、Asn-Gly/Ser/Thr(曝露区域的脱酰胺化位点)、Met(曝露区域的氧化)。将产生的人源化序列就序列相似性而在UniProtKB/Swiss-Prot数据库中blast，得出了已经作出合理假设的置信度。结果发现，所有序列都显示出与人抗体数有很高的相似性程度。此外，所有的序列都不含任何列在免疫表位数据库和分析资源(IEDB数据库)中的已知B-或T-细胞表位。The VL&VH sequence of B-B13 was blasted with the protein database (PDB) version in July 2007. Most similar light and heavy chain amino acid sequences were retrieved. The most homologous of the variable light chains was found to be 1EGJ. The most homologous of the variable heavy chains was found to be 1FNS. The 1EGJ & 1FNS structures were used to construct homology models of the variable domains, which were then energy minimized using standard procedures implemented in the Molecular Operating Environment (MOE). MOE is a comprehensive set of computer-aided drug design software provided by Chemical Computing group. Subsequently, the 3D homology model of B-B13 was calculated by molecular dynamics (MD) in Generalized Born implicit solvent (Generalized Born implicit solvent) for 1.7 nanoseconds. The resulting 1,700 snapshots of MD trajectories were then used to calculate the distribution of the root mean square difference (rmsd) for each of the B-B13 amino acids compared to the reference medoid position. Finally, a statistical test was used to compare the RMSD distribution of each amino acid with the global RMSD distribution to determine whether the amino acid was sufficiently flexible from MD calculations to be considered relevant to B cell receptors. body interaction, thereby becoming the cause of the activation of the immune response. The flexible position of the murine B-B13 variable region was compared with the position corresponding to the human antibody sequence in the ImMunoGeneTics database of the January 2007 edition downloaded to the local. Only those residues whose flexibility was greater than three times the mean and some flanking residues that preserved the 3D structure of these flexible residues were retained for the search. Select human antibody variable regions that contain the most identical flexible residues, with special consideration for those located in the CDRs positions within the range to replace flexible residues in the variable region of the murine B-B13 antibody. Some mutations in the CDRs were also included in the proposed format to avoid problematic residues. The following sequence motifs were investigated: Asp-Pro (acid labile bond), Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (deamidation site in exposed region), Met (Oxidation of exposed areas). The resulting humanized sequences were blasted in the UniProtKB/Swiss-Prot database for sequence similarity, giving a confidence level that a reasonable assumption had been made. It was found that all sequences showed a high degree of similarity to human antibody numbers. Furthermore, all sequences were free of any known B- or T-cell epitopes listed in the Immunological Epitope Database and Analysis Resource (IEDB database).
对重链提出三种形式(H1,H2,H3)并且对轻链提出三种形式(L1、L2和L3)。轻链的三种形式得自CAA83271.1(Genebank登录号CAA83271)。L1形式包含4个突变。L2形式包含一个另外的突变以除去CDR3中的DP位点(Pro99)。与L2相比，L3在CDRs中加入了两个另外的突变，它们是2个假定的脱酰胺位点(N34Q,N96A)。重链的H1,H2和H3形式得自CAC39364.1(Genebank登录号CAC39364)。该模板不是得分最高的模板，但它是不包含与已知免疫原序列显示高同源性(>70％)的得分最高的模板。H1形式包含6个突变，H2序列加入了两个另外的突变来解决三个脱酰胺位点(N60A、N73T和N83T)。氨基酸的序列编号反映了其在蛋白之中的天然次序(N端到C端)。H3包含两个另外的被认为能够提高效力的突变(Y100R&D106K)。建议6个组合的VL和VH的变体以产生人源化抗体：VL1xVH1,VL2xVH2,VL1xVH3,VL3xVH1,VL3xVH2和VL3xVH3。如表1所示，对人源化的B-B13VL和VH变体中的氨基酸利用本申请书详细说明一节中给出的表面重整技术进行了改变。左边一栏表示原始的氨基酸以及它们在鼠B-B13mAb中的位置。Three forms are proposed for the heavy chain (H1, H2, H3) and three forms for the light chain (L1, L2 and L3). The three forms of the light chain were obtained from CAA83271.1 (Genebank accession number CAA83271). The L1 form contains 4 mutations. The L2 form contains an additional mutation to remove the DP site in CDR3 (Pro99). Compared to L2, L3 incorporates two additional mutations in the CDRs, which are 2 putative deamidation sites (N34Q, N96A). The H1, H2 and H3 versions of the heavy chain were obtained from CAC39364.1 (Genebank accession number CAC39364). This template was not the highest scoring template, but it was the highest scoring template that did not contain high homology (>70%) to known immunogen sequences. The H1 form contains 6 mutations and the H2 sequence has two additional mutations added to address the three deamidation sites (N60A, N73T and N83T). The sequence numbering of amino acids reflects their natural order (N-terminal to C-terminal) within the protein. H3 contains two additional mutations (Y100R & D106K) believed to enhance potency. Six combined VL and VH variants were suggested to generate humanized antibodies: VL1xVH1, VL2xVH2, VL1xVH3, VL3xVH1, VL3xVH2 and VL3xVH3. As shown in Table 1, the amino acids in the humanized B-B13 VL and VH variants were changed using the resurfacing technique given in the detailed description section of this application. The left column shows the original amino acids and their positions in the murine B-B13 mAb.
表1Table 1
实施例4:小鼠抗人IL-4单克隆抗体克隆8D4-8的Fv结构域的人源化Example 4: Humanization of the Fv domain of mouse anti-human IL-4 monoclonal antibody clone 8D4-8
采用本文上面所述的人源化(表面重整)技术来实现8D4-8克隆的人源化。制备了两种人源化形式。一种形式在重链的CDR中包括一个被认为能解决有问题的残基(曝露的酸性不稳定位置)的突变。Humanization of the 8D4-8 clone was achieved using the humanization (resurfacing) technique described herein above. Two humanized forms were prepared. One version included a mutation in the CDR of the heavy chain that was thought to resolve the problematic residue (exposed acid-labile position).
8D4-8的VL&VH序列与2007年7月的PDB版本进行blast。检索出大多数类似的轻链和重链氨基酸序列。发现可变轻链中最同源的为1YDJ。发现可变重链中最同源的为1IQW。1YDG&1IQW结构被用来建造可变结构域的同源性模型，然后利用MOE中实施的标准程序对其进行能量最小化。其后对8D4-8的3D同源性模型在广义波恩隐含溶剂(Generalized Bornimplicit solvent)中进行了1.7纳秒的分子动力学(MD)计算。然后对每一个8D4氨基酸，利用得到的1,700个MD轨迹快照，与参照medoid位置相比较，计算其均方根差(rmsd)的分布。最后用一个统计试验对每个氨基酸的均方根差分布与全局均方根差分布进行比较，以决定该氨基酸从MD计算来看，是否有足够的柔性而有可能被视为与B细胞受体相互作用，从而成为免疫反应活化的原因。将鼠8D4-8可变区的柔性位置与下载到本地的2007年1月版ImMunoGeneTics数据库中人抗体序列对应的位置进行了比较。只有那些柔性大于平均值三倍的残基以及一些保存了这些柔性残基3D结构的侧翼残基才被保留用于搜索。选择含相同柔性残基最多的人抗体可变区，特别考虑位于CDR的范围内的位置，以便替换鼠8D4-8抗体可变区的柔性残基。最终制备一些另外的突变来避免问题残基。研究了下面的序列基序：Asp-Pro(酸性不稳定键)、Asn-X-Ser/Thr(糖基化)、Asn-Gly/Ser/Thr(曝露区域的脱酰胺化位点)、Met(曝露区域的氧化)。发现的唯一有问题的残基为重链CDR2中的DP位点。将产生的人源化序列就序列相似性而在UniProtKB/Swiss-Prot数据库中进行blast，得出了已经作出合理假设的置信度。全部序列都显示出与众多人抗体的高度相似性。此外，所有序列均不含有IEDB数据库中所列的任何已知B细胞或T细胞表位。The VL&VH sequence of 8D4-8 was blasted with the PDB version in July 2007. Most similar light and heavy chain amino acid sequences were retrieved. The most homologous of the variable light chains was found to be 1YDJ. The most homologous of the variable heavy chains was found to be 1IQW. The 1YDG & 1IQW structures were used to construct homology models of the variable domains, which were then energy minimized using standard procedures implemented in MOE. The 3D homology model of 8D4-8 was then subjected to 1.7 nanosecond molecular dynamics (MD) calculations in Generalized Bornimplicit solvent. The distribution of the root mean square difference (rmsd) was then calculated for each 8D4 amino acid using the resulting 1,700 snapshots of the MD trajectory compared to the reference medoid position. Finally, a statistical test was used to compare the rmsd distribution of each amino acid with the global rmsd distribution to determine whether the amino acid was sufficiently flexible from MD calculations to be considered relevant to B cell receptors. body interaction, thereby becoming the cause of the activation of the immune response. The flexible position of the murine 8D4-8 variable region was compared with the position corresponding to the human antibody sequence in the ImMunoGeneTics database of the January 2007 edition downloaded to the local. Only those residues whose flexibility was greater than three times the mean and some flanking residues that preserved the 3D structure of these flexible residues were retained for the search. Select human antibody variable regions that contain the most identical flexible residues, with special consideration for those located in the CDRs A range of positions to replace flexible residues in the variable region of the murine 8D4-8 antibody. Eventually some additional mutations were made to avoid problematic residues. The following sequence motifs were investigated: Asp-Pro (acid labile bond), Asn-X-Ser/Thr (glycosylation), Asn-Gly/Ser/Thr (deamidation site in exposed region), Met (Oxidation of exposed areas). The only problematic residue found was the DP site in the heavy chain CDR2. The resulting humanized sequences were blasted against the UniProtKB/Swiss-Prot database for sequence similarity, giving confidence levels that reasonable assumptions had been made. All sequences show high similarity to numerous human antibodies. Furthermore, none of the sequences contained any known B-cell or T-cell epitopes listed in the IEDB database.
提示对重链获得了两种形式(H1,H2)并且对轻链获得了一种形式(L1)。轻链的L1形式得自BAC01676.1(Genebank登录号BAC01676)。L1形式包含3个突变。重链的H1和H2形式得自BAC02418.1(Genebank登录号BAC02418)。H1形式包含9个突变，H2形式包含一个额外的突变来除去CDR2中的DP位点(Pro53)。制备了两个组合，VL1xVH1和VL1xVH2。It is suggested that two forms (H1, H2) were obtained for the heavy chain and one form (L1) for the light chain. The L1 form of the light chain was obtained from BAC01676.1 (Genebank accession number BAC01676). The L1 form contains 3 mutations. The H1 and H2 versions of the heavy chain were obtained from BAC02418.1 (Genebank accession number BAC02418). The H1 form contains 9 mutations and the H2 form contains an additional mutation to remove the DP site (Pro53) in CDR2. Two combinations were prepared, VL1xVH1 and VL1xVH2.
表2显示利用人源化(表面重整)技术对人源化的8D4-8VL和VH变体中的氨基酸所作的改变。左边一栏表示原始的氨基酸以及它们在鼠8D4-8mAb中的位置。Table 2 shows the amino acid changes in the humanized 8D4-8 VL and VH variants using humanization (resurfacing) techniques. The left column shows the original amino acids and their positions in the murine 8D4-8 mAb.
表2Table 2
实施例5:嵌合抗IL-13克隆B-B13单克隆抗体、嵌合抗IL-4克隆8D4-8单克隆抗体以及人源化变体的克隆和生成Example 5: Cloning and Generation of Chimeric Anti-IL-13 Clone B-B13 Monoclonal Antibody, Chimeric Anti-IL-4 Clone 8D4-8 Monoclonal Antibody, and Humanized Variants
抗IL-13克隆B-B13和抗IL-4克隆8D4-8的可变重链和轻链的氨基酸序列被逆转译为核苷酸序列，并用Young L.和Dong Q.(Nucl.Acids Res.(2004),32(7),e59)描述的重叠延伸PCR(OE-PCR)修改规程分别生成。PCR产物用Invitrogen TOPO TA克隆试剂盒(目录号：45-0641)被克隆到4-TOPO中，并用M13正向和M13反向引物测序。可变结构域被分别融合到恒定的重链(IGHG1，Genebank登录号Q569F4)或轻链(IGKC)Genebank登录号Q502W4)中，用NheI和HindIII消化，每个结构域被连接到Durocher等人(2002),Nucl.Acids Res.30(2),E9描述的pTT载体的类似物(analogon)游离型表达载体pXL的NheI/HindIII位点，从而产生了嵌合的B-B13重链和轻链以及嵌合的8D4-8重链和轻链的哺乳动物表达质粒。The amino acid sequences of the variable heavy and light chains of anti-IL-13 clone B-B13 and anti-IL-4 clone 8D4-8 were reverse-translated into nucleotide sequences and analyzed with Young L. and Dong Q. (Nucl.Acids Res .(2004), 32(7), e59) modified procedures for overlap extension PCR (OE-PCR) described were generated respectively. The PCR product was cloned into 4-TOPO and sequenced with M13 forward and M13 reverse primers. The variable domains were respectively fused to a constant heavy chain (IGHG1, Genebank accession number Q569F4) or light chain (IGKC) Genebank accession number Q502W4), digested with NheI and HindIII, and each domain was ligated into Durocher et al. ( 2002), Nucl.Acids Res.30(2), the analog of the pTT vector described in E9 (analogon) the NheI/HindIII site of the episomal expression vector pXL, thereby producing chimeric B-B13 heavy chain and light chain and mammalian expression plasmids for chimeric 8D4-8 heavy and light chains.
编码抗IL-13克隆B-B13和抗IL-4克隆8D4-8的人源化变体的表达克隆也透过重叠延伸PCR(OE-PCR)，基于提议的原始序列氨基酸交换合成产生。Expression clones encoding humanized variants of the anti-IL-13 clone B-B13 and the anti-IL-4 clone 8D4-8 were also generated synthetically by overlap-extension PCR (OE-PCR), based on the proposed original sequence amino acid exchange.
编码该抗体重链和轻链的表达质粒在大肠杆菌DH5a中繁殖。用于转染的质粒是利用Qiagen EndoFree Plasmid Mega试剂盒从大肠杆菌中制备的。Expression plasmids encoding the antibody heavy and light chains were propagated in E. coli DH5a. Plasmids for transfection were prepared from E. coli using the Qiagen EndoFree Plasmid Mega kit.
为转染，在500mL摇瓶里的100mL体积的无血清FreeStyle培养基(Invitrogen)中按3x 105细胞/mL接种HEK293FreeStyle细胞(Invitrogen)。细胞在一个37℃、含有8％CO2加湿空气的培养箱中培养，在以110rpm旋转的定轨摇床平台上。For transfection, HEK293 FreeStyle cells (Invitrogen) were seeded at3 x 105 cells/mL in 100 mL volumes of serum-free FreeStyle medium (Invitrogen) in 500 mL shake flasks. Cells were grown at 37°C in an incubator with 8%CO2 in humidified air on an orbital shaker platform rotating at 110 rpm.
接种三天后，利用一台CASY电子细胞计数器(System GmbH)测定了存活的细胞数和总细胞数。存活率大于90％的细胞用于转染，细胞密度为1-1.5x106细胞/mL。100mL细胞在一个有重链和轻链表达质粒混合物(5x10-7μgDNA/细胞)的500mL摇瓶里利用FugeneHD(Roche)在制备商说明的条件下进行转染，其中DNA:FugeneHD比率为2:7。转染后的细胞在一个在以110rpm旋转的定轨摇床平台上的37℃培养箱(8％CO2)中培养7天。Three days after inoculation, using a CASY electronic cell counter ( System GmbH) determined the number of viable cells and the number of total cells. Cells with greater than 90% viability were used for transfection at a cell density of1-1.5x106 cells/mL. 100 mL of cells were transfected with FugeneHD (Roche) in a 500 mL shaker flask with a mixture of heavy and light chain expression plasmids (5x10-7 μg DNA/cell) at a DNA:FugeneHD ratio of 2: 7. The transfected cells were cultured for 7 days in a 37°C incubator (8% CO2 ) on an orbital shaker platform rotating at 110 rpm.
在一个Nunc F96-MaxiSorp-Immuno板上包被山羊抗人IgG(Fc特异)[NatuTecA80-104A]。该抗体在碳酸盐包被缓冲液(50mM碳酸钠pH 9.6)中稀释到10ug/ml，每孔分配50uL。平板用胶带密封，在4℃下储存过夜。用洗涤缓冲液(PBS pH 7.4,0.1％Tween20)洗涤平板三次。每孔分配有150uL封闭溶液(1％BSA/PBS)以覆盖平板。室温下经过1小时后，用洗涤缓冲液洗三次。加入100uL样品或标准(范围为1500ng/ml到120ng/ml)，并在室温下放置1小时。用洗涤缓冲液洗平板三次。加入用孵育溶液(0.1％BSA,PBS pH 7.4,0.05％Tween20)按1:10.000稀释的100uL山羊抗人IgG-FC–HRP缀合物[NatuTec A80-104P-60]。室温下经过1小时孵育后，用洗涤缓冲液洗三次平板。向每孔中分配100uL ABTS底物(10mg ABTS片(Pierce 34026)溶于ml的0.1M Na2HPO4，0.05M柠檬酸溶液，pH 5.0。使用前加入10uL 30％H2O2/10ml底物缓冲液)，并令其显色。显色后(大约10到15分钟)，加入50uL的1％SDS溶液以终止反应。平板在A405读数。Goat anti-human IgG (Fc specific) [NatuTecA80-104A] was coated on a Nunc F96-MaxiSorp-Immuno plate. The antibody was diluted to 10 ug/ml in carbonate coating buffer (50 mM sodium carbonate pH 9.6) and 50 uL was dispensed per well. Plates were sealed with tape and stored overnight at 4°C. Plates were washed three times with wash buffer (PBS pH 7.4, 0.1% Tween20). 150 uL of blocking solution (1% BSA/PBS) was dispensed per well to cover the plate. After 1 hour at room temperature, wash three times with wash buffer. 100 uL of samples or standards (ranging from 1500 ng/ml to 120 ng/ml) were added and left at room temperature for 1 hour. Plates were washed three times with wash buffer. 100 uL goat anti-human IgG-FC-HRP conjugate [NatuTec A80-104P-60] diluted 1:10.000 with incubation solution (0.1% BSA, PBS pH 7.4, 0.05% Tween20) was added. After 1 hour incubation at room temperature, the plates were washed three times with wash buffer. Dispense 100uL of ABTS substrate (10mg ABTS tablets (Pierce 34026) in ml of 0.1MNa2HPO4 , 0.05M citricacid solution, pH 5.0 to each well. Add10uL of 30% H2O2/10ml of substrate just before use. buffer) and allowed to develop the color. After color development (approximately 10 to 15 minutes), 50 uL of 1% SDS solution was added to terminate the reaction. Plate reads at A405.
蛋白质在蛋白A(HiTrapTM Protein AHP Columns,GE Life Sciences)上以亲和色谱纯化。用100mM含有100mM NaCl的乙酸盐缓冲液pH3.5洗脱后，单克隆抗体配制在PBS溶液中并经过0.22μm过滤。蛋白质浓度通过测量在280nm的吸光度而测定。每一批用Protein200Plus LabChip试剂盒在Agilent 2100生物分析仪上在还原和非还原条件下进行分析，以测定每个亚单位和单体的纯度和分子量。Proteins were purified by affinity chromatography on Protein A (HiTrap™ Protein AHP Columns , GE Life Sciences). After elution with 100 mM acetate buffer pH 3.5 containing 100 mM NaCl, the monoclonal antibodies were formulated in PBS and filtered through 0.22 μm. Protein concentration was determined by measuring absorbance at 280 nm. Each batch was analyzed with the Protein200Plus LabChip kit on an Agilent 2100 Bioanalyzer under reducing and non-reducing conditions to determine the purity and molecular weight of each subunit and monomer.
实施例6:人源化抗IL-13克隆B-B13变异体和人源化抗IL-4克隆8D4-8变异体的表征Example 6: Characterization of humanized anti-IL-13 clone B-B13 variant and humanized anti-IL-4 clone 8D4-8 variant
重组人IL-13和IL-4试剂购自Chemicon(USA)。按下述方法进行了Biacore动力学分析。Recombinant human IL-13 and IL-4 reagents were purchased from Chemicon (USA). Biacore kinetic analysis was performed as follows.
利用表面等离振子技术在一台Biacore 3000(GE Healthcare)上对纯化的抗体进行了详细的动力学表征。采用了一种捕获测定，其利用一种物种特异的抗体(例如人-Fc特异性MAB 1302,Chemicon)对感兴趣的抗体进行捕获和定向。捕获抗体用标准程序通过伯胺(10000RU)固定在研究级CM5芯片(GE Life Science)上。捕获所分析的抗体并调节RU值，导致在流速为10μl/min时得到最大的30RU分析物结合。在0到50nM之间的HBS EP浓度范围(10mM HEPES pH 7.4,150mM NaCl,3mM EDTA,0.005％表面活性剂P20)，流速为30μl/min时，测量相对于重组IL-4和IL-13的结合动力学。用10mM甘氨酸pH2.5再生芯片表面。利用没有捕获的抗体的流动池作为参照，在BIA评估计划包中对动力学参数进行了分析和计算。为了研究两个抗原的加成结合，应用一个向导驱动(wizard-driven)的共注射方法，其中一个抗原注射后，立即注射IL-13/IL-4的抗原混合物。The purified antibodies were subjected to detailed kinetic characterization using surface plasmon technology on a Biacore 3000 (GE Healthcare). A capture assay is employed that utilizes a species-specific antibody (eg, human-Fc specific MAB 1302, Chemicon) to capture and orient the antibody of interest. Capture antibodies were immobilized on research grade CM5 chips (GE Life Science) via primary amines (10000 RU) using standard procedures. The analyzed antibody was captured and the RU value was adjusted to result in a maximum of 30 RU analyte binding at a flow rate of 10 μl/min. Relative to recombinant IL-4 and IL-13 was measured at a concentration range of HBS EP between 0 and 50 nM (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant P20) at a flow rate of 30 μl/min Binding Kinetics. The chip surface was regenerated with 10 mM glycine pH 2.5. Kinetic parameters were analyzed and calculated in the BIA Evaluation Plan package using the flow cell without captured antibody as a reference. To study the additive binding of two antigens, a wizard-driven co-injection method was applied, where one antigen was injected immediately followed by an antigen mixture of IL-13/IL-4.
籍由测量TF-1细胞中IL-4或IL-13介导的细胞增殖的抑制，测量了本发明抗体的生物活性。简而言之，申请人用IL-4或IL-13来刺激TF-1细胞的生长。TF-1是一个生长依赖于细胞因子并对很多细胞因子，包括IL-4和IL-13有反应的细胞系。诱导的生长(与不存在细胞因子时的基线条件相比)则代表了IL-4或IL-13的生物活性。抗IL-4、抗IL-13以及双特异的抗IL-4/IL-13抗体被表明阻断IL-4或IL-13诱导的TF-1细胞生长。此外，双特异的抗IL-4/IL-13抗体已被表明阻断由组合的IL-4和IL-13刺激所诱导的TF-1细胞增殖。阻断效应按剂量依赖方式测量以产生IC50(50％抑制时的抗体浓度)作为抗体对其靶，即IL-4或IL-13的中和能力。下面更详细地介绍所采用的方法细节。The biological activity of the antibodies of the invention was measured by measuring the inhibition of IL-4 or IL-13 mediated cell proliferation in TF-1 cells. Briefly, Applicants used IL-4 or IL-13 to stimulate the growth of TF-1 cells. TF-1 is a cytokine-dependent cell line for growth and responsive to many cytokines, including IL-4 and IL-13. Induced growth (compared to baseline conditions in the absence of cytokines) is representative of IL-4 or IL-13 biological activity. Anti-IL-4, anti-IL-13, and bispecific anti-IL-4/IL-13 antibodies were shown to block IL-4 or IL-13-induced growth of TF-1 cells. Furthermore, bispecific anti-IL-4/IL-13 antibodies have been shown to block TF-1 cell proliferation induced by combined IL-4 and IL-13 stimulation. The blocking effect was measured in a dose-dependent manner to yield IC50 (antibody concentration at 50% inhibition) as the ability of the antibody to neutralize its target, IL-4 or IL-13. Details of the methodology employed are presented in more detail below.
TF-1细胞(ATCC,CRL-2003)被保持在含有新加入hGM-CSF(最终浓度为4ng/ml)的完全培养基(含有高葡萄糖、25mM Hepes缓冲液和谷氨酰胺、10％FBS、1x P/S、1mM丙酮酸钠的DMEM)中。IL-13(15ng/ml)或IL-4(1ng/ml)处理前24小时。细胞在96孔板上按0.05x106/ml接种在不含hGM-CSF的完全培养基中。在加入到细胞之前，在37℃预温育系列稀释的具有对应细胞因子的抗体30分钟。细胞培养了72小时(37℃、5％CO2)。加入了cellTiter96Aqueous的MTS/PMS溶液。然后孵育细胞3小时。该阶段后，在490nm利用一个培养板读数器记录吸光度。用Speed软件计算了IC50值。TF-1 cells (ATCC, CRL-2003) were maintained in complete medium (containing high glucose, 25 mM Hepes buffer and glutamine, 10% FBS, 1x P/S, 1 mM sodium pyruvate in DMEM). 24 hours before IL-13 (15 ng/ml) or IL-4 (1 ng/ml) treatment. Cells were seeded on 96-well plates at 0.05x106/ml in complete medium without hGM-CSF. Serially diluted antibodies with the corresponding cytokines were pre-incubated at 37°C for 30 minutes before adding to the cells. Cells were cultured for 72 hours (37°C, 5% CO2). Add the MTS/PMS solution of cellTiter96Aqueous. Cells were then incubated for 3 hours. After this period, absorbance was recorded at 490 nm using a plate reader. IC50 values were calculated with Speed software.
人源化B-B13变异体的结合动力学和中和活性示于表3中(nt、未测)。The binding kinetics and neutralizing activity of the humanized B-B13 variants are shown in Table 3 (nt, not tested).
表3table 3
与原来的鼠B-B13(13倍)和嵌合B-B13(6倍)相比，一种人源化的B-B13变异体，huB-B13VL3xVH2，具有显著更高的亲和力。当这些人源化的抗IL-13抗体用于治疗哮喘患者时，改善的亲和力可导致效能和效力的提高。此外，用于人时，与鼠抗体或嵌合抗体相比，人源化抗体可具有降低的免疫原性。A humanized B-B13 variant, huB-B13VL3xVH2, had significantly higher affinity than original murine B-B13 (13-fold) and chimeric B-B13 (6-fold). The improved affinity can lead to increased potency and efficacy when these humanized anti-IL-13 antibodies are used to treat asthmatic patients. Furthermore, humanized antibodies may have reduced immunogenicity when used in humans compared to murine or chimeric antibodies.
人源化8D4-8变异体的结合动力学和中和活性示于表4中。The binding kinetics and neutralizing activity of the humanized 8D4-8 variants are shown in Table 4.
表4Table 4
与原来的鼠8D4-8(11倍)和嵌合8D4-8(2倍)相比，一个人源化的8D4-8变异体，hu8D4-8VL1xVH1，具有显著更高的亲和力。当这个人源化的抗IL-4抗体用于治疗哮喘患者时，改善的亲和力可导致效能和效力的提高。此外，用于人时，与鼠抗体或嵌合抗体相比，人源化抗体可具有降低的免疫原性。A humanized 8D4-8 variant, hu8D4-8VL1xVH1 , had significantly higher affinity than the original murine 8D4-8 (11-fold) and chimeric 8D4-8 (2-fold). The improved affinity can lead to increased potency and potency when this humanized anti-IL-4 antibody is used to treat asthmatic patients. Furthermore, humanized antibodies may have reduced immunogenicity when used in humans compared to murine or chimeric antibodies.
实施例7：人源化抗IL-4/IL-13双特异抗体的克隆和生成Example 7: Cloning and production of humanized anti-IL-4/IL-13 bispecific antibody
用于表达双特异抗体(BsAb)的形式是US5,989,830描述的双结构域双头形式的IgG变体。这一形式中，IgG分子在其相应的重链和轻链的N端被第二个抗体的另外一个可变结构域延伸。因此，得到的IgG分子是个由两个重链和两个轻链组成的异四聚体。重链由两个可变的重结构域(VH1-VH2)组成，来自由连接体连接在一起的两个不同的抗体，所述连接体由十个氨基酸(G4S)2组成，并且被融合到IgG4恒定结构域。轻链由两个可变的轻域(VL1-VL2)组成，来自由连接体连接在一起两个不同抗体，所述连接体由十个氨基酸(G4S)2组成，并被融合到恒定κ区域。The format used to express bispecific antibodies (BsAbs) is the IgG variant of the two-domain double-head format described in US 5,989,830. In this format, the IgG molecules are extended at the N-terminus of their respective heavy and light chains by an additional variable domain of a second antibody. Thus, the resulting IgG molecule is a heterotetramer composed of two heavy chains and two light chains. The heavy chain consists of two variable heavy domains (VH1-VH2) from two different antibodies joined together by a linker consisting of ten amino acids (G4S)2 fused to IgG4 constant domain. The light chain consists of two variable light domains (VL1-VL2) from two different antibodies joined together by a linker consisting of ten amino acids (G4S)2 fused to a constant kappa region .
8D4-8变体的可变重结构域和轻结构域的序列是由PCR产生的，在其各自的编码(G4S)2-(GGA TCC)-8D4-8之一部份的5’-端引入了BamHI限制位点(GGA TCC)。8D4-8人源化变异体VH的3’序列以ApaI限制位点(编码CH1结构域的第一个氨基酸)结束，用于后面融合到IGHG4序列(缺失了末端Lys并带有S241P和L248E双突变的Q569F4)。VL8D4-8的3’-端以编码恒定κ链的前两个氨基酸的BsiWI限制位点结束，用于后面融合到IGKC(Genebank登录号Q502W4)。The sequences of the variable heavy and light domains of the 8D4-8 variant were generated by PCR at the 5'-end of their respective parts encoding (G4S)2 -(GGA TCC )-8D4-8 A BamHI restriction site (GGA TCC ) was introduced. The 3' sequence of the 8D4-8 humanized variant VH ends with an ApaI restriction site (encoding the first amino acid of the CH1 domain) for subsequent fusion to the IGHG4 sequence (deleted terminal Lys with S241P and L248E double mutant Q569F4). The 3'-end of VL8D4-8 ends with a BsiWI restriction site encoding the first two amino acids of the constant kappa chain for subsequent fusion to IGKC (Genebank accession number Q502W4).
B-B13变体的可变重结构域和轻结构域的序列是由PCR产生的，在其各自的编码(G4S)2-(B-B13)-(GGA GGC GGA GGG TCC GGA GGC GGAGGA TCC(SEQ ID NO:7))之一部份的3’-端引入了BamHI限制位点。B-B13变异体的VH和VL序列是由NheI限制位点在其各自的5’-端产生的，接着是ATG起始密码子和编码序列的前导肽。The sequences of the variable heavy and light domains of the B-B13 variant were generated by PCR in their respective codes (G4S)2 -(B-B13)-(GGA GGC GGA GGG TCC GGA GGC GGAGGA TCC (SEQ ID NO:7)) a BamHI restriction site was introduced into the 3'-end of a part. The VH and VL sequences of the B-B13 variant were generated by NheI restriction sites at their respective 5'-ends, followed by the ATG start codon and the leader peptide of the coding sequence.
B-B13和8D4-8的VH透过它们的BamHI位点在(G4S)2连接体内被融合到一起。B-B13和8D4-8的VL透过它们的BamHI位点在(G4S)2连接体内被彼此融合到。因此，产生的重链和轻链的串联具有以下组成。The VHs of B-B13 and 8D4-8 were fused together within the (G4S)2 linker through their BamHI sites. The VLs of B-B13 and 8D4-8 were fused to each other within the (G4S)2 linker through their BamHI sites. Thus, the resulting concatenation of heavy and light chains has the following composition.
双特异抗体重链：NheI-前导肽-VH-B-B13-(G4S)2-VH 8D4-8-ApaI。Bispecific antibody heavy chain: NheI-leader peptide-VH-B-B13-(G4S)2 -VH 8D4-8-ApaI.
双特异抗体轻链：NheI-前导肽-VL-B-B13-(G4S)2-VL 8D4-8-BsiWI。Bispecific antibody light chain: NheI-leader peptide-VL-B-B13-(G4S)2 -VL 8D4-8-BsiWI.
所有PCR中间片段用Invitrogen TOPO TA克隆试剂盒(目录号：45-0641)克隆到4-TOPO中，并用M13正向和M13反向引物进行测序。All PCR intermediate fragments were cloned into 4-TOPO and sequenced with M13 forward and M13 reverse primers.
序列确认后，重链串联通过其ApaI位点融合到IGHG4序列，可变的轻链串联透过其BsiWI位点融合到IGKC。产生的双结构域重链和轻链用NheI和HindIII消化，每个被连接到游离型基因表达载体pXL的NheI/HindIII位点中，分别产生了用于TBTI重链和轻链哺乳动物表达的质粒。After sequence confirmation, the heavy chain tandem was fused to the IGHG4 sequence through its ApaI site, and the variable light chain tandem was fused to IGKC through its BsiWI site. The resulting dual-domain heavy and light chains were digested with NheI and HindIII, and each was ligated into the NheI/HindIII sites of the episomal gene expression vector pXL to generate TBTI heavy and light chain mammalian expression, respectively. plasmid.
根据下面表5所示的B-B13和8D4-8的人源化VH和VL形式的组合，产生了四个人源化双特异抗IL-4/抗IL-13构建体。According to the combination of the humanized VH and VL forms of B-B13 and 8D4-8 shown in Table 5 below, four humanized bispecific anti-IL-4/anti-IL-13 constructs were generated.
表5table 5
实施例8：人源化双特异抗体的表征Example 8: Characterization of humanized bispecific antibodies
结合和中活性测定按上面实施例中的说明进行。Binding and neutralization assays were performed as described in the Examples above.
表6给出了四个人源化的抗IL-4/IL-13抗体变异体的结合动力学。所有四个双特异抗体构建体均以高亲和力结合到IL-4和IL-13。Table 6 presents the binding kinetics of the four humanized anti-IL-4/IL-13 antibody variants. All four bispecific antibody constructs bound to IL-4 and IL-13 with high affinity.
表6Table 6
人源化抗IL-4/IL-13双特异抗体变异体的中和活性总结于表7中。huTBTI3-1_1和huTBTI3-2_1完全中和了IL-13或IL-4诱导的TF-1细胞增殖，IC50如下所示。The neutralizing activities of the humanized anti-IL-4/IL-13 bispecific antibody variants are summarized in Table 7. huTBTI3-1_1 and huTBTI3-2_1 completely neutralized IL-13 or IL-4-induced proliferation of TF-1 cells with IC50 as shown below.
表7Table 7
众所周知，突变的IL-13等位基因高频率的与哮喘相关联(Heinzmann A.等人,2000,Hum Mol Genet 9,4,第549-559页)。因此，研究了双特异抗体对突变的IL-13蛋白质(人IL-13R112Q变体，PeproTech,Rocky Hill,NJ,USA)的结合动力学。结果表明，huTBTI3-1_1和huTBTI3-2_1与IL-13变异体的结合和与野生型IL-13的结合类似。It is well known that a high frequency of mutated IL-13 alleles is associated with asthma (Heinzmann A. et al., 2000, Hum Mol Genet 9, 4, pp. 549-559). Therefore, the binding kinetics of bispecific antibodies to mutated IL-13 protein (human IL-13R112Q variant, PeproTech, Rocky Hill, NJ, USA) was studied. The results showed that huTBTI3-1_1 and huTBTI3-2_1 bound IL-13 variants similarly to wild-type IL-13.
表8显示人源化抗IL-4/IL-13分子与突变的IL-13蛋白质的结合动力学。Table 8 shows the binding kinetics of humanized anti-IL-4/IL-13 molecules to mutated IL-13 proteins.
表8Table 8
序列表sequence listing
<110> 塞诺菲-安万特股份有限公司(sanofi-aventis US Inc)<110> sanofi-aventis US Inc
E·劳(Rao, Ercole)E. Law (Rao, Ercole)
V·米科尔(Mikol, Vincent)V. Mikol (Mikol, Vincent)
D·李(Li, Danxi)D. Li (Li, Danxi)
J·克鲁伊普(Kruip, Jochen)J. Kruip (Kruip, Jochen)
M·戴维森(Davison, Matthew)M. Davison (Davison, Matthew)
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Gln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala TyrGln Gly Arg Ala Thr Leu Thr Val Asp Glu Ser Thr Ser Thr Ala Tyr
65 70 75 8065 70 75 80
Met Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr CysMet Gln Leu Arg Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95 85 90 95
Thr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp ValThr Arg Leu Lys Glu Tyr Gly Asn Tyr Asp Ser Phe Tyr Phe Asp Val
100 105 110 100 105 110
Trp Gly Ala Gly Thr Leu Val Thr Val Ser Ser AlaTrp Gly Ala Gly Thr Leu Val Thr Val Ser Ser Ser Ala
115 120 115 120
<210> 6<210> 6
<211> 10<211> 10
<212> PRT<212> PRT
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 连接体<223> Connector
<400> 6<400> 6
Gly Gly Gly Gly Ser Gly Gly Gly Gly SerGly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 101 5 10
<210> 7<210> 7
<211> 30<211> 30
<212> DNA<212>DNA
<213> 人工序列<213> Artificial sequence
<220><220>
<223> 引物<223> Primer
<400> 7<400> 7
ggaggcggag ggtccggagg cggaggatcc 30ggaggcggag ggtccggagg cggaggatcc 30
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| Application Number | Title | Priority Date | Filing Date |
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| CN200880111668.6ADivisionCN101827863B (en) | 2007-10-15 | 2008-10-14 | Antibodies binding to IL‑4 and/or IL‑13 and uses thereof |
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| CN106986938Atrue CN106986938A (en) | 2017-07-28 |
| CN106986938B CN106986938B (en) | 2021-07-09 |
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| CN202110668094.XAActiveCN113480647B (en) | 2007-10-15 | 2008-10-14 | Antibodies binding to IL-4 and/or IL-13 and uses thereof |
| CN200880111668.6AActiveCN101827863B (en) | 2007-10-15 | 2008-10-14 | Antibodies binding to IL‑4 and/or IL‑13 and uses thereof |
| CN201710131539.4AActiveCN106986938B (en) | 2007-10-15 | 2008-10-14 | Antibodies that bind to IL-4 and/or IL-13 and uses thereof |
| Application Number | Title | Priority Date | Filing Date |
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| CN202110668094.XAActiveCN113480647B (en) | 2007-10-15 | 2008-10-14 | Antibodies binding to IL-4 and/or IL-13 and uses thereof |
| CN200880111668.6AActiveCN101827863B (en) | 2007-10-15 | 2008-10-14 | Antibodies binding to IL‑4 and/or IL‑13 and uses thereof |
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